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Sample records for adenylyl cyclase sac

  1. pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)

    PubMed Central

    Rahman, Nawreen; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3′, 5′ monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO−3) ions. In living cells, HCO−3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO−3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role. PMID:24324443

  2. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals

    PubMed Central

    Tresguerres, Martin; Barott, Katie L.; Barron, Megan E.; Roa, Jinae N.

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3−, and sAC has been confirmed to be a HCO3− sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3−-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  3. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals.

    PubMed

    Tresguerres, Martin; Barott, Katie L; Barron, Megan E; Roa, Jinae N

    2014-03-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3(-), and sAC has been confirmed to be a HCO3(-) sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3(-)-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H(+) absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  4. Soluble Adenylyl Cyclase in Health and Disease

    PubMed Central

    Schmid, Andreas; Meili, Dimirela; Salathe, Matthias

    2014-01-01

    The second messenger cAMP is integral for many physiological processes. Soluble adenylyl cyclase (sAC) was recently identified as a widely expressed intracellular source of cAMP in mammalian cells. sAC is evolutionary, structurally, and biochemically distinct from the G-protein-responsive transmembranous adenylyl cyclases (tmAC). The structure of the catalytic unit of sAC is similar to tmAC, but sAC does not contain transmembranous domains, allowing localizations independent of the membranous compartment. sAC activity is stimulated by HCO3-, Ca2+ and is sensitive to physiologically relevant ATP fluctuations. sAC functions as a physiological sensor for carbon dioxide and bicarbonate, and therefore indirectly for pH. Here we review the physiological role of sAC in different human tissues with a major focus on the lung. PMID:25064591

  5. Adenylyl cyclases in the digestive system.

    PubMed

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2014-06-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed.

  6. Soluble adenylyl cyclase is essential for proper lysosomal acidification.

    PubMed

    Rahman, Nawreen; Ramos-Espiritu, Lavoisier; Milner, Teresa A; Buck, Jochen; Levin, Lonny R

    2016-10-01

    Lysosomes, the degradative organelles of the endocytic and autophagic pathways, function at an acidic pH. Lysosomes are acidified by the proton-pumping vacuolar ATPase (V-ATPase), but the molecular processes that set the organelle's pH are not completely understood. In particular, pH-sensitive signaling enzymes that can regulate lysosomal acidification in steady-state physiological conditions have yet to be identified. Soluble adenylyl cyclase (sAC) is a widely expressed source of cAMP that serves as a physiological pH sensor in cells. For example, in proton-secreting epithelial cells, sAC is responsible for pH-dependent translocation of V-ATPase to the luminal surface. Here we show genetically and pharmacologically that sAC is also essential for lysosomal acidification. In the absence of sAC, V-ATPase does not properly localize to lysosomes, lysosomes fail to fully acidify, lysosomal degradative capacity is diminished, and autophagolysosomes accumulate. PMID:27670898

  7. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  8. CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

    PubMed

    Zippin, Jonathan H; Chen, Yanqiu; Straub, Susanne G; Hess, Kenneth C; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G; Sharp, Geoffrey W G; Levin, Lonny R; Buck, Jochen

    2013-11-15

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

  9. AKAPs and Adenylyl Cyclase in Cardiovascular Physiology and Pathology

    PubMed Central

    Efendiev, Riad; Dessauer, Carmen W.

    2011-01-01

    Cyclic AMP, generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins (AKAPs) localize the effect of cAMP in space and time by organizing receptors, adenylyl cyclase, protein kinase A and other components of the cAMP cascade into multiprotein complexes. In this review we discuss how interaction of AKAPs with distinct AC isoforms affects cardiovascular physiology. PMID:21978991

  10. Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

    PubMed

    Nunes, Ana R; Holmes, Andrew P S; Sample, Vedangi; Kumar, Prem; Cann, Martin J; Monteiro, Emília C; Zhang, Jin; Gauda, Estelle B

    2013-08-15

    Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

  11. Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

    PubMed Central

    Kolodecik, Thomas R.; Shugrue, Christine A.; Thrower, Edwin C.; Levin, Lonny R.; Buck, Jochen; Gorelick, Fred S.

    2012-01-01

    An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis. PMID:22844459

  12. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

    Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments. PMID:12940771

  13. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    SciTech Connect

    Smith, Natasha; Kim, Sook-Kyung; Reddy, Prasad T.; Gallagher, D. Travis

    2006-03-01

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V{sub M} = 3.0 Å{sup 3} Da{sup −1}. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

  14. Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

    PubMed

    Witters, Erwin; Quanten, Lieve; Bloemen, Jo; Valcke, Roland; Van Onckelen, Harry

    2004-01-01

    In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

  15. Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase

    SciTech Connect

    Villacres, E.C.; Xia, Z.; Bookbinder, L.H.; Edelhoff, S.; Disteche, C.M.; Storm, D.R.

    1993-05-01

    The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3{prime} end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5{prime} half of the bovine type I adenylyl cyclase and the 3{prime} half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7. 36 refs., 4 figs.

  16. Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation

    PubMed Central

    Shaw, Peter X.; Fang, Jiahua; Sang, Alan; Wang, Yan; Kapiloff, Michael S.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development. Methods Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type–specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice. Results Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice. Conclusions sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors. PMID:27679853

  17. Control of outflow resistance by soluble adenylyl cyclase.

    PubMed

    Lee, Yong Suk; Marmorstein, Alan D

    2014-01-01

    Abstract Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure, even in patients with normal tension glaucoma. Typically, this is accomplished by reducing the rate of aqueous flow by limiting aqueous production or enhancing drainage using drugs and surgery. Whereas these strategies are effective in diminishing vision loss, some patients continue to lose vision and many discontinue use of their medications because of undesirable side effects. Drugs known to be effective in altering conventional outflow have for the most part been abandoned from modern clinical practice due to undesirable side effects. Identification of new drugs that could enhance conventional outflow, would offer additional options in the treatment of glaucoma and ocular hypertension. To this end, our laboratory has recently uncovered a novel pathway for regulation of conventional outflow by the ciliary body. This pathway is dependent on soluble adenylyl cyclase, an enzyme that catalyzes the generation of cyclic adenosine 3',5' monophosphate (cAMP) in response to bicarbonate.

  18. Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    PubMed Central

    Fenton, Robert A.; Murray, Fiona; Dominguez Rieg, Jessica A.; Tang, Tong; Levi, Moshe

    2014-01-01

    Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) enhance phosphate excretion by the proximal tubule of the kidney by retrieval of the sodium-dependent phosphate transporters (Npt2a and Npt2c) from the apical plasma membrane. PTH activates adenylyl cyclase (AC) through PTH 1 receptors and stimulates the cAMP/PKA signaling pathway. However, the precise role and isoform(s) of AC in phosphate homeostasis are not known. We report here that mice lacking AC6 (AC6−/−) have increased plasma PTH and FGF-23 levels compared with wild-type (WT) mice but comparable plasma phosphate concentrations. Acute activation of the calcium-sensing receptor or feeding a zero phosphate diet almost completely suppressed plasma PTH levels in both AC6−/− and WT mice, indicating a secondary cause for hyperparathyroidism. Pharmacologic blockade of FGF receptors resulted in a comparable increase in plasma phosphate between genotypes, whereas urinary phosphate remained significantly higher in AC6−/− mice. Compared with WT mice, AC6−/− mice had reduced renal Npt2a and Npt2c protein abundance, with approximately 80% of Npt2a residing in lysosomes. WT mice responded to exogenous PTH with redistribution of Npt2a from proximal tubule microvilli to intracellular compartments and lysosomes alongside a PTH-induced dose–response relationship for fractional phosphate excretion and urinary cAMP excretion. These responses were absent in AC6−/− mice. In conclusion, AC6 in the proximal tubule modulates cAMP formation, Npt2a trafficking, and urinary phosphate excretion, which are highlighted by renal phosphate wasting in AC6−/− mice. PMID:24854272

  19. Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c.

    PubMed

    Sinha, Sangita C; Wetterer, Martina; Sprang, Stephen R; Schultz, Joachim E; Linder, Jürgen U

    2005-02-23

    Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site. PMID:15678099

  20. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  1. [Reactivity of the adenylyl cyclase system in rat tissues to biogenic amines and peptide hormones under starvation condition].

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-04-01

    Under starvation condition, sensitivity of the adenylyl cyclase system to regulatory action of biogenic amines and peptide hormones in rat tissues are changed. In the myocardium and skeletal muscles, after 2 and 4 days of starvation, the regulatory effects of isoproterenol and relaxin acting via G,-proteins on the adenylyl cyclase activity and the G-protein GTP-binding are significantly increased compared with control. At the same time, regulatory effects ofsomatostatin which are realized via Gi-proteins, on adenylyl cyclase system in the myocardium are decreased. Under prolonged starvation consisting of two consecutive 4-days periods, the effects of hormones acting via Gs-proteins on the adenylyl cyclase activity in muscle tissues are decreased to control value levels. The effects of hormones acting via Gi-proteins are largely reduced. In the brain, intensification of adenylyl cyclase stimulating hormonal effects was late and only observed after a 4-day starvation. Unlike muscle tissues, the increase of adenylyl cyclase stimulating effects in the brain is preserved after two-period starvation. The weakening of adenylyl cyclase inhibiting hormonal signals both in the brain and muscles is observed after a 2-day starvation and then the weakening is intensified. Possible role of glucose level and basal adenylyl cyclase activity in determination of the sensitivity of the adenylyl cyclase system to hormones under study is discussed. It is suggested that one of the key causes of physiological changes in animal organism under starvation involves alteration of hormonal signalling systems sensitivity, in particular that of the adenylyl cyclase system, to hormone regulatory action.

  2. DIFERENTIALLY EXPRESSED ADENYLYL CYCLASE ISOFORMS MEDIATE SECRETORY FUNCTIONS IN CHOLANGIOCYTE SUBPOPULATION

    PubMed Central

    Strazzabosco, Mario; Fiorotto, Romina; Melero, Saida; Glaser, Shannon; Francis, Heather; Spirlì, Carlo; Alpini, Gianfranco

    2009-01-01

    cAMP is generated by adenylyl cyclases (ACs) a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli and are differentially modulated in cholestasis. Methods: Small (SDC) and large (LDC) cholangiocytes were isolated from controls and from lipopolysaccharide-treated (LPS) or α-naphthylisothiocyanate-treated (ANIT) rats. ACs isoforms expression was assessed by real-time PCR. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO3−/CO2, cholinergic and β-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or sAC with siRNA. Results: Gene expression of the Ca2+-insensitive isoforms (AC4, AC7) was higher in SDC, while that of the Ca2+-inhibitable (AC5, AC6, AC9), the Ca2+/calmodulin stimulated AC8, and the soluble sAC, was higher in LDC. Ca2+/calmodulin-inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin-inibitors was cAMP-dependent and GABA-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO3−]i, was cAMP-dependent and inhibited by sAC-inhibitior and by sAC gene silencing. Treatment with LPS or ANIT increased expression of AC7 and sAC, while decreasing that of the others ACs. Conclusion: These studies demonstrate a previously unrecognized role of AC in biliary pathophysiology. In fact: 1) ACs isoforms are differentially expressed in cholangiocyte subpopulations, 2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, β-adrenergic agonists, or changes in [HCO3−]i, respectively, 3) ACs gene expression is modulated in experimental cholestasis. PMID:19444869

  3. Regional distribution of somatostatin receptor binding and modulation of adenylyl cyclase activity in Alzheimer's disease brain.

    PubMed

    Bergström, L; Garlind, A; Nilsson, L; Alafuzoff, I; Fowler, C J; Winblad, B; Cowburn, R F

    1991-10-01

    We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder. PMID:1684616

  4. Opposing effects of ethanol on pig ovarian adenylyl cyclase desensitized by human choriogonadotropin or isoproterenol.

    PubMed

    Ekstrom, R C; Hunzicker-Dunn, M

    1990-11-01

    Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid

  5. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  6. Overexpression of the type 1 adenylyl cyclase in the forebrain leads to deficits of behavioral inhibition.

    PubMed

    Chen, Xuanmao; Cao, Hong; Saraf, Amit; Zweifel, Larry S; Storm, Daniel R

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition.

  7. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light. PMID:24201148

  8. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

    PubMed Central

    Premont, R T; Chen, J; Ma, H W; Ponnapalli, M; Iyengar, R

    1992-01-01

    cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission. Images PMID:1409703

  9. H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway.

    PubMed

    Li, Sen; Ping, Na-Na; Cao, Lei; Mi, Yan-Ni; Cao, Yong-Xiao

    2015-12-15

    Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca(2+) channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

  10. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

  11. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.

  12. Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis.

    PubMed

    Stepanovic, Vesna; Wessels, Deborah; Daniels, Karla; Loomis, William F; Soll, David R

    2005-04-01

    Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.

  13. Role of adenylyl cyclase in reduced β-adrenoceptor-mediated vasorelaxation during maturation

    PubMed Central

    López-Canales, O.A.; Castillo-Hernandez, M.C.; Vargas-Robles, H.; Rios, A.; López-Canales, J.S.; Escalante, B.

    2016-01-01

    Beta-adrenergic receptor (βAR)-dependent blood vessel relaxation is impaired in older animals and G protein activation has been suggested as the causative mechanism. Here, we investigated the role of βAR subtypes (β1AR, β2AR, and β3AR) and cAMP in maturation-dependent vasorelaxation impairment. Aortic rings from 15 Sprague-Dawley male rats (3 or 9 weeks old) were harvested and left intact or denuded of the endothelium. Vascular relaxation in aortic rings from younger and older groups was compared in the presence of βAR subtype agonists and antagonists along with cAMP and cGMP antagonists. Isolated aortic rings were used to evaluate relaxation responses, protein expression was evaluated by western blot or real time PCR, and metabolites were measured by ELISA. Expression of βAR subtypes and adenylyl cyclase was assessed, and cAMP activity was measured in vascular tissue from both groups. Isoproterenol- and BRL744-dependent relaxation in aortic rings with and without endothelium from 9-week-old rats was impaired compared with younger rats. The β1AR antagonist CGP20712A (10-7 M) did not affect isoproterenol or BRL744-dependent relaxation in arteries from either group. The β2AR antagonist ICI-118,551 (10-7 M) inhibited isoproterenol-dependent aortic relaxation in both groups. The β3AR antagonist SR59230A (10-7 M) inhibited isoproterenol- and BRL744-dependent aortic ring relaxation in younger but not in older rats. All βAR subtypes were expressed in both groups, although β3AR expression was lower in the older group. Adenylyl cyclase (SQ 22536) or protein kinase A (H89) inhibitors prevented isoproterenol-induced relaxation in younger but not in older rats. Production of cAMP was reduced in the older group. Adenylyl cyclase III and RyR3 protein expression was higher in the younger group. In conclusion, altered expression of β3AR and adenylyl cyclase III may be responsible for reduced cAMP production in the older group. PMID:27383122

  14. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    PubMed

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  15. Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni.

    PubMed

    Hildebrandt, J D; Codina, J; Risinger, R; Birnbaumer, L

    1984-02-25

    The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This "5K peptide" is also present in preparations of another protein which we termed "40K protein" on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type. PMID:6321456

  16. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    PubMed Central

    Tong, Tao; Shen, Ying; Lee, Han-Woong; Yu, Rina; Park, Taesun

    2016-01-01

    Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity. PMID:27678003

  17. Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

    PubMed

    Adeniran, A J; Shoshani, I; Minuth, M; Awad, J A; Elce, J S; Johnson, R A

    1995-03-01

    We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin. PMID:7756444

  18. Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.

    PubMed

    Ng, G Y; George, S R; Zastawny, R L; Caron, M; Bouvier, M; Dennis, M; O'Dowd, B F

    1993-11-01

    Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Novel metabotropic glutamate receptor negatively coupled to adenylyl cyclase in cultured rat cerebellar astrocytes.

    PubMed

    Kanumilli, Srinivasan; Toms, Nick J; Roberts, Peter J

    2004-04-01

    Several excitatory amino acid ligands were found potently to inhibit forskolin-stimulated cAMP accumulation in rat cultured cerebellar astrocytes: L-cysteine sulfinic acid (L-CSA) = L-aspartate > L-glutamate >/= the glutamate uptake inhibitor, L-PDC. This property did not reflect activation of conventional glutamate receptors, since the selective ionotropic glutamate receptor agonists NMDA, AMPA, and kainate, as well as several mGlu receptor agonists [(1S,3R)-ACPD, (S)-DHPG, DCG-IV, L-AP4, L-quisqualate, and L-CCG-I], were without activity. In addition, the mGlu receptor antagonists, L-AP3, (S)-4CPG, Eglu, LY341495, (RS)-CPPG, and (S)-MCPG failed to reverse 30 microM glutamate-mediated inhibitory responses. L-PDC-mediated inhibition was abolished by the addition of the enzyme glutamate-pyruvate transaminase. This finding suggests that the effect of L-PDC is indirect and that it is mediated through endogenously released L-glutamate. Interestingly, L-glutamate-mediated inhibitory responses were resistant to pertussis toxin, suggesting that G(i)/G(o) type G proteins were not involved. However, inhibition of protein kinase C (PKC, either via the selective PKC inhibitor GF109203X or chronic PMA treatment) augmented glutamate-mediated inhibitory responses. Although mGlu3 receptors (which are negatively coupled to adenylyl cyclase) are expressed in astrocyte populations, in our study Western blot analysis indicated that this receptor type was not expressed in cerebellar astrocytes. We therefore suggest that cerebellar astrocytes express a novel mGlu receptor, which is negatively coupled to adenylyl cyclase, and possesses an atypical pharmacological profile. PMID:14999808

  20. Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulation.

    PubMed

    Carvalho, Edison S M; Gregório, Sílvia F; Power, Deborah M; Canário, Adelino V M; Fuentes, Juan

    2012-12-01

    In the marine fish intestine luminal, HCO₃⁻ can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO₃⁻ secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH-Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 μM FK + 500 μM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO₃⁻ results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 μM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen. PMID:22752677

  1. Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulation.

    PubMed

    Carvalho, Edison S M; Gregório, Sílvia F; Power, Deborah M; Canário, Adelino V M; Fuentes, Juan

    2012-12-01

    In the marine fish intestine luminal, HCO₃⁻ can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO₃⁻ secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH-Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 μM FK + 500 μM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO₃⁻ results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 μM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen.

  2. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain.

    PubMed

    Wang, Hansen; Xu, Hui; Wu, Long-Jun; Kim, Susan S; Chen, Tao; Koga, Kohei; Descalzi, Giannina; Gong, Bo; Vadakkan, Kunjumon I; Zhang, Xuehan; Kaang, Bong-Kiun; Zhuo, Min

    2011-01-12

    Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury-induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.

  3. Inhibitory role of monovalent ions on rat brain cortex adenylyl cyclase activity.

    PubMed

    Nikolic, Ivana; Mitrovic, Marina; Zelen, Ivanka; Zaric, Milan; Kastratovic, Tatjana; Stanojevic, Marijana; Nenadovic, Milutin; Stojanovic, Tomislav

    2013-10-01

    Adenylyl cyclases, comprise of a large family of enzymes that catalyze synthesis of the cyclic AMP from ATP. The aim of our study was to determine the effect of monovalent ions on both basal, stimulated adenylate cyclase EC 4.6.1.1 (AC) activity and C unit of AC and on GTPase active G-protein in the synaptic membranes of rat brain cortex. The effect of ion concentration from 30 to 200 mM (1 mM MgCl2) showed dose-dependent and significant inhibition of the basal AC activity, stimulated and unstimulated C unit activity. Stimulation of AC with 5 μM GTPγS in the presence of 50-200 mM of tested salts showed inhibitory effect on the AC activity. From our results it could be postulated that the investigated monovalent ions exert inhibitory effect on the AC complex activity by affecting the intermolecular interaction of the activated α subunit of G/F protein and the C unit of AC complex an inhibitory influence of tested monovalent ions on these molecular interaction.

  4. Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

    PubMed Central

    Beste, Kerstin Y.; Spangler, Corinna M.; Burhenne, Heike; Koch, Karl-Wilhelm; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2013-01-01

    Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides. PMID:23922959

  5. Soluble adenylyl cyclase is not required for axon guidance to netrin-1.

    PubMed

    Moore, Simon W; Lai Wing Sun, Karen; Xie, Fang; Barker, Philip A; Conti, Marco; Kennedy, Timothy E

    2008-04-01

    During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1. PMID:18400890

  6. Persistent Electrical Activity in Primary Nociceptors after Spinal Cord Injury Is Maintained by Scaffolded Adenylyl Cyclase and Protein Kinase A and Is Associated with Altered Adenylyl Cyclase Regulation

    PubMed Central

    Bavencoffe, Alexis; Li, Yong; Wu, Zizhen; Yang, Qing; Herrera, Juan; Kennedy, Eileen J.

    2016-01-01

    Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. Persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) has been found to make major contributions to chronic pain in a rat model of spinal cord injury (SCI) (Bedi et al., 2010; Yang et al., 2014). The occurrence of SCI-induced SA in a large fraction of DRG neurons and the persistence of this SA long after dissociation of the neurons provide an opportunity to define intrinsic cell signaling mechanisms that chronically drive SA in pain pathways. The present study demonstrates that SCI-induced SA requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of AC. AC activity stimulated by Ca2+-calmodulin increased, while the inhibition of AC activity by Gαi showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons. SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major clinical problem with poorly understood mechanisms and inadequate treatments. Recent findings indicate that chronic pain in a rat SCI model depends upon hyperactivity in dorsal root ganglia (DRG) neurons. Although cAMP signaling is involved in many forms of neural plasticity, including hypersensitivity of nociceptors in the presence of inflammatory mediators, our finding that continuing cAMP-PKA signaling is required for persistent SA months after SCI and long after isolation of

  7. Protein-Protein Docking and Analysis Reveal That Two Homologous Bacterial Adenylyl Cyclase Toxins Interact with Calmodulin Differently*S⃞

    PubMed Central

    Guo, Qing; Jureller, Justin E.; Warren, Julia T.; Solomaha, Elena; Florián, Jan; Tang, Wei-Jen

    2008-01-01

    Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly (∼200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a β-hairpin region (amino acids 259–273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein. PMID:18583346

  8. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. PMID:11533056

  9. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

  10. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.

  11. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein.

    PubMed

    Vojtek, A B; Cooper, J A

    1993-07-01

    CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

  12. Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding.

    PubMed

    Levy, Daniel; Marlowe, Charles; Kane-Maguire, Kim; Bao, Ming; Cherbavaz, Diana; Tomlinson, James; Sedlock, David; Scarborough, Robert

    2002-11-01

    The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme. PMID:12372507

  13. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    PubMed

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  14. Ontogeny of regulatory mechanisms for beta-adrenoceptor control of rat cardiac adenylyl cyclase: targeting of G-proteins and the cyclase catalytic subunit.

    PubMed

    Zeiders, J L; Seidler, F J; Slotkin, T A

    1997-02-01

    Fetal and neonatal tissues are resistant to catecholamine-induced desensitization of essential physiological responses. We examined the mechanisms underlying the ontogeny of desensitization in neonatal rat heart for the beta-adrenergic receptor/adenylyl cyclase signaling cascade. Animals of different ages received isoproterenol daily or 4 days and cardiac membrane preparations were evaluated on the 5th day (6, 15, 25 days old and adults). Measurements were made of basal activity, activity stimulated by two agonists (isoproterenol or glucagon) that operate at different receptors but that share Gs as the transduction intermediate, or by forskolin-Mn' to assess total catalytic capacity of the cyclase subunit; we also assessed inhibition of activity by carbachol which acts via muscarinic cholinergic receptors and G. Adult rats exhibited robust desensitization of the adenylyl cyclase response but the effect was heterologous in that equivalent loss of activity was seen for basal, isoproterenol- and glucagon-stimulated activity forskolin-Mn(2+)-stimulated activity was also decreased. Two factors contributed to desensitization; generalized reduction in membrane protein concentrations caused by cell enlargement (reduced surface-to-volume ratio), and specific interference with the G-protein component that couples receptors to the cyclase. Thus, after adjustment for changes in membrane protein, the desensitization of the forskolin-Mn2, response was no longer evident, but the effects on the other measures were still present. In addition, isoproterenol treatment produced crosstalk with the carbachol/Gi signaling pathway, with significant reductions in the ability of carbachol to inhibit adenylyl cyclase activity. Heterologous desensitization by isoproterenol was also present in 15 and 25 day old rats, but involved only selective components of the effects seen in adults. At 25 days, uncoupling of signals operating through Gs and Gi was obtained without a reduction in forskolin

  15. A Chemoattractant-mediated Gi-coupled Pathway Activates Adenylyl Cyclase in Human Neutrophils

    PubMed Central

    Mahadeo, Dana C.; Janka-Junttila, Mirkka; Smoot, Rory L.; Roselova, Pavla

    2007-01-01

    Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Gαs-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Gαs-meditated pathway. PMID:17135293

  16. Laboratory evolution of adenylyl cyclase independent learning in Drosophila and missing heritability.

    PubMed

    Cressy, M; Valente, D; Altick, A; Kockenmeister, E; Honegger, K; Qin, H; Mitra, P P; Dubnau, J

    2014-07-01

    Gene interactions are acknowledged to be a likely source of missing heritability in large-scale genetic studies of complex neurological phenotypes. However, involvement of rare variants, de novo mutations, genetic lesions that are not easily detected with commonly used methods and epigenetic factors also are possible explanations. We used a laboratory evolution study to investigate the modulatory effects of background genetic variation on the phenotypic effect size of a null mutation with known impact on olfactory learning. To accomplish this, we first established a population that contained variation at just 23 loci and used selection to evolve suppression of the learning defect seen with null mutations in the rutabaga adenylyl cyclase. We thus biased the system to favor relatively simplified outcomes by choosing a Mendelian trait and by restricting the genetic variation segregating in the population. This experimental design also assures that the causal effects are among the known 23 segregating loci. We observe a robust response to selection that requires the presence of the 23 variants. Analyses of the underlying genotypes showed that interactions between more than two loci are likely to be involved in explaining the selection response, with implications for the missing heritability problem.

  17. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

    PubMed Central

    Zhou, Zhiwen; Tanaka, Kenji F.; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  18. Type 3 adenylyl cyclase: a key enzyme mediating the cAMP signaling in neuronal cilia

    PubMed Central

    Qiu, Liyan; LeBel, Robert P; Storm, Daniel R; Chen, Xuanmao

    2016-01-01

    Cilia are rigid, centriole-derived, microtubule-based organelles present in a majority of vertebrate cells including neurons. They are considered the cellular “antennae” attuned for detecting a range of extracellular signals including photons, odorants, morphogens, hormones and mechanical forces. The ciliary microenvironment is distinct from most actin-based subcellular structures such as microvilli or synapses. In the nervous system, there is no evidence that neuronal cilia process any synaptic structure. Apparently, the structural features of neuronal cilia do not allow them to harbor any synaptic connections. Nevertheless, a large number of G protein-coupled receptors (GPCRs) including odorant receptors, rhodopsin, Smoothened, and type 6 serotonin receptor are found in cilia, suggesting that these tiny processes largely depend on metabotropic receptors and their tuned signals to impact neuronal functions. The type 3 adenylyl cyclase (AC3), widely known as a cilia marker, is highly and predominantly expressed in olfactory sensory cilia and primary cilia throughout the brain. We discovered that ablation of AC3 in mice leads to pleiotropic phenotypes including anosmia, failure to detect mechanical stimulation of airflow, cognitive deficit, obesity, and depression-like behaviors. Multiple lines of human genetic evidence also demonstrate that AC3 is associated with obesity, major depressive disorder (MDD), sarcoidosis, and infertility, underscoring its functional importance. Here we review recent progress on AC3, a key enzyme mediating the cAMP signaling in neuronal cilia. PMID:27785336

  19. Structure of the Class IV Adenylyl Cyclase Reveals a Novel Fold

    SciTech Connect

    Gallagher,D.; Smith, N.; Kim, S.; Heroux, A.; Robinson, H.; Reddy, P.

    2006-01-01

    The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 {angstrom} resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.

  20. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

    PubMed

    Zhou, Zhiwen; Tanaka, Kenji F; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  1. In silico prediction of tyrosinase and adenylyl cyclase inhibitors from natural compounds.

    PubMed

    Fong, Pedro; Tong, Henry H Y; Chao, Chi M

    2014-02-01

    Although many herbal medicines are effective in the treatment of hyperpigmentation, the potency of different constituents remains unknown. In this work, more than 20,000 herbal ingredients from 453 herbs were docked into the crystal structures of adenylyl cyclase and a human homology tyrosinase model using Surflex-Dock. These two enzymes are responsible for melanin production and inhibition of them may attain a skin-whitening effect superior to currently available agents. The essential drug properties for topical formulation of the herbal ingredients, including skin permeability, sensitization, irritation, corrosive and carcinogenic properties were predicted by Dermwin, Skin Sensitization Alerts (SSA), Skin Irritation Corrosion Rules Estimation Tool (SICRET) and Benigni/Bossa rulebase module of Toxtree. Moreover, similarity ensemble and pharmacophore mapping approaches were used to forecast other potential targets for these herbal compounds by the software, SEArch and PharmMapper. Overall, this study predicted seven compounds to have advanced drug-like properties over the well-known effective tyrosinase inhibitors, arbutin and kojic acid. These seven compounds have the highest potential for further in vitro and in vivo investigation with the aim of developing safe and high-efficacy skin-whitening agents.

  2. The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG.

    PubMed

    Pedroza-Roldán, César; Aceves-Sánchez, Michel de Jesús; Zaveri, Anisha; Charles-Niño, Claudia; Elizondo-Quiroga, Darwin Eduardo; Hernández-Gutiérrez, Rodolfo; Allen, Kirk; Visweswariah, Sandhya S; Flores-Valdez, Mario Alberto

    2015-01-01

    All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

  3. Adenylyl Cyclase Plays a Regulatory Role in Development, Stress Resistance and Secondary Metabolism in Fusarium fujikuroi

    PubMed Central

    García-Martínez, Jorge; Ádám, Attila L.; Avalos, Javier

    2012-01-01

    The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H2O2), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group. PMID:22291883

  4. Type VI adenylyl cyclase negatively regulates GluN2B-mediated LTD and spatial reversal learning

    PubMed Central

    Chang, Ching-Pang; Lee, Cheng-Ta; Hou, Wen-Hsien; Lin, Meng-Syuan; Lai, Hsing-Lin; Chien, Chen-Li; Chang, Chen; Cheng, Pei-Lin; Lien, Cheng-Chang; Chern, Yijuang

    2016-01-01

    The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca2+ and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6−/− mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus. PMID:26932446

  5. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  6. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    PubMed

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  7. Effects of abstinence and family history for alcoholism on platelet adenylyl cyclase activity.

    PubMed

    Menninger, J A; Barón, A E; Tabakoff, B

    1998-12-01

    Platelet adenylyl cyclase (AC) activity was measured in 32 alcohol-dependent subjects and 27 control subjects who were categorized as either family history-positive (FHP) or family history-negative (FHN) for alcoholism. The interview and blood sample collections were performed shortly after cessation of heavy drinking in the alcoholic group, and repeat blood samples were obtained at the end of the first and second weeks of monitored abstinence. Control subjects received the same interview and provided blood samples at the time of the interview. When subjects were not segregated for FHP or FHN status, there were no statistically significant differences in basal, cesium fluoride (CsF)-, or forskolin-stimulated mean AC activities between the controls and the alcoholics, at study entry or with 1 or 2 weeks of abstinence. On the other hand, over the 2-week course of sobriety from heavy drinking, the CsF-stimulated AC activity of FHP alcohol-dependent subjects decreased significantly (p = 0.03). FHP alcohol-dependent subjects after 2 weeks of sobriety had significantly lower mean CsF-stimulated AC activity than FHN controls (p = 0.04), whereas the FHN alcoholic subjects' CsF-stimulated AC activity did not differ significantly from FHN controls at this point in time. When all subjects were pooled and then categorized as either FHP or FHN, there was a significant difference in mean CsF-stimulated AC activity (p = 0.02) between the FHP and FHN subject groups. Genetic factors and abstinence appear to have roles in determining low platelet AC activity in alcoholic and nonalcoholic subjects. CsF-stimulated platelet AC activity, in particular, appears to act as a trait marker for a genetic vulnerability to developing alcoholism, but recent heavy drinking in male alcoholics is a factor that can mask differences between FHP and FHN subjects.

  8. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  9. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  10. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice.

    PubMed

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R; Ma, Runlin Z

    2015-11-30

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3(-/-)) and wild-type (AC3(+/+)) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3(-/-) mice was significantly altered, compared to AC3(+/+) mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3(-/-) and AC3(+/+) mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

  11. Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model

    PubMed Central

    Kang, Wen-bo; Yang, Qi; Guo, Yan-yan; Wang, Lu; Wang, Dong-sheng; Cheng, Qiang; Li, Xiao-ming; Tang, Jun; Zhao, Jian-ning; Liu, Gang; Zhuo, Min

    2016-01-01

    Background Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. Results Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. Conclusions NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex. PMID:27612915

  12. Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

    PubMed Central

    Rodriguez-Centeno, Javier; Sastre, Leandro

    2016-01-01

    Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation. PMID:26840347

  13. The role of adenylyl cyclase isoform 6 in β-adrenoceptor signalling in murine airways

    PubMed Central

    Birrell, Mark A; Bonvini, Sara J; Wortley, Michael A; Buckley, James; Yew-Booth, Liang; Maher, Sarah A; Dale, Nicole; Dubuis, Eric D; Belvisi, Maria G

    2015-01-01

    BACKGROUND AND PURPOSE Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β-adrenoceptor agonists. EXPERIMENTAL APPROACH Receptors mediating responses to β-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls. KEY RESULTS Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to β-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished. CONCLUSIONS AND IMPLICATIONS AC6 played a critical role in relaxation of ASM to β1-adrenoceptor agonists and in modulation of sensory nerves by β1-3-adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine. PMID:25205328

  14. Differential Interactions of the Catalytic Subunits of Adenylyl Cyclase with Forskolin Analogs

    PubMed Central

    Pinto, Cibele; Hübner, Melanie; Gille, Andreas; Richter, Mark; Mou, Tung-Chung; Sprang, Stephen R.; Seifert, Roland

    2009-01-01

    The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I–VIII. Diterpenes without C1-OH group do not activate ACs. The C1-OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′-O-(N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS ~ 7-deacetyl-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS > 1-deoxy-FS ~ 1,9-dideoxy-FS ~ 7-deacetyl-1-deoxy-FS ~ 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS > 7-deacety-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS ≫ 1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C1-OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2. PMID:19447224

  15. [Spatial memory and regulation of brain adenylyl cyclase by serotonin and dopamine in rat with streptozotocin diabetes].

    PubMed

    Sukhov, I B; Chistyakova, O V; Shipilov, V N; Doilnitsyn, A M; Shpakov, A O

    2015-03-01

    The most common complication of diabetes mellitus of the type 1 (DM1) is a cognitive deficiency, which develops as a result of neurodegenerative changes in the brain. The aim of this work was to study the learning and spatial memory in rats with streptozotocin DM1 with different duration (1.5 and 6 months), as well as the activity of adenylyl cyclase signaling system (ACSS) sensitive to agonists of the serotonin and the dopamine receptors in the brain of diabetic rats. Our experiments demonstrated that rats with 1.5-months DM1 has no changes in spatial memory which were evaluated in a Morris water maze whereas in rats with 6-months DM1 the spatial memory and learning ability were decreased. The alterations of the regulation of adenylyl cyclase by agonists of types 1 and 6 serotonin receptors and type 2 dopamine receptors were found in both the 1.5- and 6-months DM1 which indicates their importance in the development of cognitive deficiency. Abnormalities in the. brain ACSS can be considered as key factors in the etiology and pathogenesis of cognitive dysfunctions in DM1. Hypothesized that cognitive deficiency occurs only in the later stages of DM1 due to alterations in the serotonin and dopamine signaling systems of the brain.

  16. All five cloned human somatostatin receptors (hSSTR1-5) are functionally coupled to adenylyl cyclase.

    PubMed

    Patel, Y C; Greenwood, M T; Warszynska, A; Panetta, R; Srikant, C B

    1994-01-28

    Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

  17. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

    PubMed

    Hess, Kenneth C; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A; Buck, Jochen; Levin, Lonny R; Barrientos, Antoni

    2014-10-01

    Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

  18. Impairment of adenylyl cyclase-mediated glutamatergic synaptic plasticity in the periaqueductal grey in a rat model of neuropathic pain

    PubMed Central

    Ho, Yu-Cheng; Cheng, Jen-Kun; Chiou, Lih-Chu

    2015-01-01

    Key points Long-lasting neuropathic pain has been attributed to elevated neuronal plasticity changes in spinal, peripheral and cortical levels. Here, we found that reduced neuronal plasticity in the ventrolateral periaqueductal grey (vlPAG), a midbrain region important for initiating descending pain inhibition, may also contribute to neuropathic pain. Forskolin- and isoproterenol (isoprenaline)-elicited EPSC potentiation was impaired in the vlPAG of a rat model of neuropathic pain induced by spinal nerve injury. Down-regulation of adenylyl cyclase–cAMP– PKA signalling, due to impaired adenylyl cyclase, but not phosphodiesterase, in glutamatergic terminals may contribute to the hypofunction of excitatory synaptic plasticity in the vlPAG of neuropathic rats and the subsequent descending pain inhibition, ultimately leading to long-lasting neuropathic pain. Our results suggest that drugs that activate adenylyl cyclase in the vlPAG have the potential for relieving neuropathic pain. Abstract Neuropathic pain has been attributed to nerve injury-induced elevation of peripheral neuronal discharges and spinal excitatory synaptic plasticity while little is known about the contribution of neuroplasticity changes in the brainstem. Here, we examined synaptic plasticity changes in the ventrolateral (vl) periaqueductal grey (PAG), a crucial midbrain region for initiating descending pain inhibition, in spinal nerve ligation (SNL)-induced neuropathic rats. In vlPAG slices of sham-operated rats, forskolin, an adenylyl cyclase (AC) activator, produced long-lasting enhancement of EPSCs. This is a presynaptic effect since forskolin decreased the paired-pulse ratio and failure rate of EPSCs, and increased the frequency, but not the amplitude, of miniature EPSCs. Forskolin-induced EPSC potentiation was mimicked by a β-adrenergic agonist (isoproterenol (isoprenaline)), and prevented by an AC inhibitor (SQ 22536) and a cAMP-dependent protein kinase (PKA) inhibitor (H89), but not by a

  19. Mice Overexpressing Type 1 Adenylyl Cyclase Show Enhanced Spatial Memory Flexibility in the Absence of Intact Synaptic Long-Term Depression

    ERIC Educational Resources Information Center

    Zhang, Ming; Wang, Hongbing

    2013-01-01

    There is significant interest in understanding the contribution of intracellular signaling and synaptic substrates to memory flexibility, which involves new learning and suppression of obsolete memory. Here, we report that enhancement of Ca[superscript 2+]-stimulated cAMP signaling by overexpressing type 1 adenylyl cyclase (AC1) facilitated…

  20. Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter.

    PubMed

    Banjac, Ana; Zimmermann, Markus O; Boeckler, Frank M; Kurz, Ursula; Schultz, Anita; Schultz, Joachim E

    2012-03-01

    The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly. Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.

  1. Studies of the molecular mechanisms of action of relaxin on the adenylyl cyclase signaling system using synthetic peptides derived from the LGR7 relaxin receptor.

    PubMed

    Shpakov, A O; Gur'yanov, I A; Kuznetsova, L A; Plesneva, S A; Shpakova, E A; Vlasov, G P; Pertseva, M N

    2007-09-01

    The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619-629, 619-629-Lys(Palm), and 615-629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619-629-Lys(Palm) and 615-629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385-394 of the alpha(s) subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with G(s) protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the G(s) protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C

  2. High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role

    PubMed Central

    Barott, K. L.; Helman, Y.; Haramaty, L.; Barron, M. E.; Hess, K. C.; Buck, J.; Levin, L. R.; Tresguerres, M.

    2013-01-01

    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels. PMID:23459251

  3. Hydroxamate based inhibitors of adenylyl cyclase. Part 2: the effect of cyclic linkers on P-site binding.

    PubMed

    Levy, Daniel; Bao, Ming; Tomlinson, James; Scarborough, Robert

    2002-11-01

    The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored. PMID:12372508

  4. Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8-deficient mice.

    PubMed

    Bosse, Kelly E; Charlton, Jennifer L; Susick, Laura L; Newman, Brooke; Eagle, Andrew L; Mathews, Tiffany A; Perrine, Shane A; Conti, Alana C

    2015-12-01

    The cAMP/protein kinase A pathway regulates methamphetamine (METH)-induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild-type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH-induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)-evoked accumbal dialysate dopamine levels were similar between genotypes, saline-treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH-sensitized DKO mice compared to saline-treated controls, an effect not observed in WT mice. Notably, saline-treated DKO mice had significantly increased phosphorylated Dopamine- and cAMP-regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug-induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin-stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine-induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP-32 activation. These findings indicate AC 1/8 signaling

  5. Block by gabapentin of the facilitation of glutamate release from rat trigeminal nucleus following activation of protein kinase C or adenylyl cyclase

    PubMed Central

    Maneuf, Yannick P; McKnight, Alexander T

    2001-01-01

    The effect of activation of protein kinase C (PKC) or adenylyl cyclase on release of glutamate has been investigated in a perfused slice preparation from the rat caudal trigeminal nucleus. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) produced a concentration-dependent increase in K+-evoked release of [2H]-glutamate (maximum increase 45%, EC50 11.8 nM), but in the presence of gabapentin (30 μM) the facilitation of release was blocked. The adenylyl cyclase activator forskolin (FSK) also induced a concentration-dependent increase in K+-evoked release of [3H]-glutamate (maximum increase 36%, EC50 2.4 μM), and again this facilitatory effect was blocked by gabapentin (30 μM). We suggest that these results may be of relevance to the antihyperalgesic properties of gabapentin, in conditions where concomitant release of substance P and CGRP produces activation of PKC and adenylyl cyclase respectively. PMID:11564640

  6. Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2014-01-01

    Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes the plant cell death by the inhibiting glutamine synthetase, which leads to an abnormal accumulation of ammonium ions. To better understand the molecular mechanisms involved in TβL-induced cell death and necrotic wildfire lesions, we focused on adenylyl cyclase in Nicotiana benthamiana. We isolated the gene designated as NbAC (Nicotiana benthamiana adenylyl cyclase). Recombinant NbAC protein showed adenylyl cyclase activity in vitro. TβL-induced necrotic lesions were significantly suppressed in NbAC-silenced leaves compared with control plant leaves. However, the amount of ammonium ions was scarcely affected by NbAC-silencing. Furthermore, the silencing of NbAC also suppressed l-methionine sulfoximine-induced cell death without any changes in the amount of ammonium accumulated. When inoculated directly with P. syringae pv tabaci, NbAC-silenced plants showed reduced symptoms. These results suggest that NbAC might play an essential role in intracellular signal transduction during TβL-induced cell death and necrotic wildfire disease development.

  7. The catalytic domains of thiamine triphosphatase and CyaB-like adenylyl cyclase define a novel superfamily of domains that bind organic phosphates

    PubMed Central

    Iyer, Lakshminarayan M; Aravind, L

    2002-01-01

    Background The CyaB protein from Aeromonas hydrophila has been shown to possess adenylyl cyclase activity. While orthologs of this enzyme have been found in some bacteria and archaea, it shows no detectable relationship to the classical nucleotide cyclases. Furthermore, the actual biological functions of these proteins are not clearly understood because they are also present in organisms in which there is no evidence for cyclic nucleotide signaling. Results We show that the CyaB like adenylyl cyclase and the mammalian thiamine triphosphatases define a novel superfamily of catalytic domains called the CYTH domain that is present in all three superkingdoms of life. Using multiple alignments and secondary structure predictions, we define the catalytic core of these enzymes to contain a novel α+β scaffold with 6 conserved acidic residues and 4 basic residues. Using contextual information obtained from the analysis of gene neighborhoods and domain fusions, we predict that members of this superfamily may play a central role in the interface between nucleotide and polyphosphate metabolism. Additionally, based on contextual information, we identify a novel domain (called CHAD) that is predicted to functionally interact with the CYTH domain-containing enzymes in bacteria and archaea. The CHAD is predicted to be an alpha helical domain, and contains conserved histidines that may be critical for its function. Conclusions The phyletic distribution of the CYTH domain suggests that it is an ancient enzymatic domain that was present in the Last Universal Common Ancestor and was involved in nucleotide or organic phosphate metabolism. Based on the conservation of catalytic residues, we predict that CYTH domains are likely to chelate two divalent cations, and exhibit a reaction mechanism that is dependent on two metal ions, analogous to nucleotide cyclases, polymerases and certain phosphoesterases. Our analysis also suggests that the experimentally characterized members of this

  8. Pharmacological stimulation of type 5 adenylyl cyclase stabilizes heart rate under both microgravity and hypergravity induced by parabolic flight.

    PubMed

    Bai, Yunzhe; Tsunematsu, Takashi; Jiao, Qibin; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Jin, Meihua; Cai, Wenqian; Jin, Hui-Ling; Fujita, Takayuki; Ichikawa, Yasuhiro; Suita, Kenji; Kurotani, Reiko; Yokoyama, Utako; Sato, Motohiko; Iwatsubo, Kousaku; Ishikawa, Yoshihiro; Okumura, Satoshi

    2012-01-01

    We previously demonstrated that type 5 adenylyl cyclase (AC5) functions in autonomic regulation in the heart. Based on that work, we hypothesized that pharmacological modulation of AC5 activity could regulate the autonomic control of the heart rate under micro- and hypergravity. To test this hypothesis, we selected the approach of activating AC5 activity in mice with a selective AC5 activator (NKH477) or inhibitor (vidarabine) and examining heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater under micro- and hypergravity in the vidarabine group, while there were no significant changes in the NKH477 group, suggesting that autonomic regulation was unstable in the vidarabine group. The ratio of low frequency and high frequency (HF) in heart rate variability analysis, a marker of sympathetic activity, became significantly decreased under micro- and hypergravity in the NKH477 group, while there was no such decrease in the vidarabine group. Normalized HF, a marker of parasympathetic activity, became significantly greater under micro- and hypergravity in the NKH477 group. In contrast, there was no such increase in the vidarabine group. This study is the first to indicate that pharmacological modulation of AC5 activity under micro- and hypergravity could be useful to regulate the autonomic control of the heart rate.

  9. Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

    SciTech Connect

    Cauwe, Benedicte; Martens, Erik; Van den Steen, Philippe E.; Proost, Paul; Van Aelst, Ilse; Blockmans, Daniel; Opdenakker, Ghislain

    2008-09-10

    Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1 was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.

  10. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  11. Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

    PubMed

    Mogensen, Estelle Geweiss; Janbon, Guilhem; Chaloupka, James; Steegborn, Clemens; Fu, Man Shun; Moyrand, Frédérique; Klengel, Torsten; Pearson, David S; Geeves, Michael A; Buck, Jochen; Levin, Lonny R; Mühlschlegel, Fritz A

    2006-01-01

    Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH. PMID:16400172

  12. Effect of overexpressed adenylyl cyclase VI on β1- and β2-adrenoceptor responses in adult rat ventricular myocytes

    PubMed Central

    Stark, Joalice C C; Haydock, Stephen F; Foo, Roger; Brown, Morris J; Harding, Sian E

    2004-01-01

    Adenylyl cyclase VI (ACVI) is one of the most abundantly expressed β adrenergic receptor (βAR)-coupled cyclases responsible for cyclic AMP (cAMP) production within the mammalian myocardium. We investigated the role of ACVI in the regulation of cardiomyocyte contractility and whether it is functionally coupled with β1 adrenergic receptor (β1AR). Recombinant adenoviruses were generated for ACVI and for antisense to ACVI (AS). Adult rat ventricular myocytes were transfected with ACVI virus, AS or both (SAS). Adenovirus for green fluorescent protein (GFP) served as control. Myocyte contraction amplitudes (% shortening) and relaxation times (R50) were analysed. ACVI function was determined using cAMP assays. ACVI-transfected cells demonstrated a strong 139 kDa ACVI protein band compared to controls. ACVI myocytes had higher steady-state intracellular cAMP levels than GFP myocytes when unstimulated (GFP vs ACVI=6.60±0.98 vs 14.2±2.1 fmol cAMP/viable cell, n=4, P<0.05) and in the presence of 1 μM isoprenaline or 10 μM forskolin. ACVI myocytes had increased basal contraction (% shortening: GFP vs ACVI: 1.90±1.36 vs 3.91±2.29, P<0.0001) and decreased basal R50 (GFP vs ACVI: 62.6±24.2 ms (n=50) vs 45.0±17.2 ms (n=248), P<0.0001). ACVI myocyte responses were increased for forskolin (Emax: GFP=6.70±1.59 (n=6); ACVI=9.06±0.69 (n=14), P<0.01) but not isoprenaline. ACVI myocyte responses were increased (Emax: GFP vs ACVI=3.16±0.77 vs 5.10±0.60, P<0.0001) to xamoterol (a partial β1AR-selective agonist) under β2AR blockade (+50 nM ICI 118, 551). AS decreased both control and ACVI-stimulated xamoterol responses (Emax: AS=2.59±1.42, SAS=1.38±0.5). ACVI response was not mimicked by IBMX. Conversely, response through β2 adrenergic receptor (β2AR) was decreased in ACVI myocytes. In conclusion, ACVI overexpression constitutively increases myocyte contraction amplitudes by raising cAMP levels. Native ACVI did not contribute to basal cAMP production or contraction

  13. Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Seifert, Roland

    2015-01-01

    Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca2+-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca2+-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism. PMID:25946093

  14. Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia

    PubMed Central

    Guadiana, Sarah M.; Parker, Alexander K.; Filho, Gileno F.; Sequeira, Ashton; Semple-Rowland, Susan; Shaw, Gerry; Mandel, Ronald J.; Foster, Thomas C.; Kumar, Ashok; Sarkisian, Matthew R.

    2016-01-01

    The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain

  15. Clozapine effects on adenylyl cyclase activity and serotonin type 1A receptors in human brain post-mortem.

    PubMed

    Marazziti, Donatella; Baroni, Stefano; Palego, Lionella; Betti, Laura; Giannaccini, Gino; Castagna, Maura; Naccarato, Antonio G; Luccachini, Antonio; Catena-Dell'Osso, Mario; Dell'Osso, Liliana

    2014-04-01

    Although the pharmacological profile of the atypical antipsychotic clozapine has been extensively studied in animal models, little information is available on its effects in the human brain. In particular, much interest is focused on the understanding of clozapine activity on serotonin (5-HT) neurotransmission, particularly on 5-HT receptor of type 1A (5-HT(1A)) that seems to play a pivotal role in the control of the 5-HT system. The present work, therefore, aimed at evaluating the effects of clozapine and its major metabolite, norclozapine, on the modulation of adenylyl cyclase (AC) velocity via 5-HT(1A) receptors in human post-mortem brain regions, in particular the prefrontal cortex, hippocampus and raphe nuclei. Concomitantly, the ability of the two compounds to displace the specific binding of the 5-HT(1A) receptor agonist [³H]-8-hydroxy-(2-di-N-propylamino) tetralin ([³H]-8-OH-DPAT) was evaluated in the same brain areas. The results showed that both clozapine and norclozapine, although with a 20-fold lower affinity, displaced [³H]8-OH-DPAT binding in all of the brain regions analysed, suggesting their interaction with 5-HT(1A) receptors. At the same time, clozapine and, to a lesser extent, norclozapine were found to inhibit the forskolin (FK)-stimulated AC system, while decreasing cyclic adenosine monophosphate (cAMP) concentrations in the hippocampus only. The receptor characterisation of the clozapine effect on AC observed in the hippocampus by the use of antagonists showed a mixed profile, involving not only the 5-HT(1A) receptor but also a muscarinic (M) receptor subtype, most likely the M₄ one. These findings, while considering all the limitations due to the use of post-mortem tissues, are strongly suggestive of a region-dependent pharmacological action of clozapine in the human brain that may explain its peculiar clinical effects and open up research towards novel targets for future antipsychotic drugs.

  16. Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock

    PubMed Central

    Chen, Dong; Buchanan, Gordon F.; Ding, Jian M.; Hannibal, Jens; Gillette, Martha U.

    1999-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light. PMID:10557344

  17. Inhibition of adenylyl cyclase in amygdala blocks the effect of audiogenic seizure kindling in genetically epilepsy-prone rats.

    PubMed

    Tupal, Srinivasan; Faingold, Carl

    2010-01-01

    Genetically epilepsy-prone rats of the severe seizure strain (GEPR-9s) exhibit audiogenic seizures (AGS) beginning with wild running and ending with tonic hind limb extension (TE). AGS kindling in GEPR-9s involves periodic repetition of >/=14 seizures over 7-21 days and results in prolonged seizures and an additional phase of generalized post-tonic clonus (PTC) that follows TE. AGS kindling behavior changes are long-lasting and involve expansion of the requisite seizure neuronal network from the brainstem to include the amygdala, mediated by neuroplasticity in lateral amygdala. Recent evidence indicates that focal activation of adenylyl cyclase (AC) in lateral amygdala leads to precipitous acquisition of AGS-kindled seizure behaviors, suggesting that activation of AC activity is important in development and maintenance of AGS kindling. The present study further examined the role of AC in AGS-kindled seizures in GEPR-9s by focally inhibiting AC in the amygdala. Bilateral microinjection of an AC inhibitor, SQ22,536 (0.25 and 0.50 nmol/side), in AGS-kindled GEPR-9s selectively blocked PTC during AGS at 1 h after microinjection, but the pre-kindled AGS behaviors remained intact. The incidence of PTC during AGS returned to pre-drug levels 12 h after the lower dose of SQ22,536 (0.25 nmol/side). However, after the higher dose of SQ22,536 (0.5 nmol/side), complete return to AGS with PTC was seen in all GEPR-9s at 120 h. These results indicate that maintenance of AGS kindling-mediated PTC in GEPR-9s may involve activation of AC. These data provide further evidence for the involvement of AC in the epileptogenic mechanisms subserving AGS kindling.

  18. Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization.

    PubMed Central

    Ng, G Y; Trogadis, J; Stevens, J; Bouvier, M; O'Dowd, B F; George, S R

    1995-01-01

    The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation. Images Fig. 2 Fig. 3 PMID:7479745

  19. Antiviral drug vidarabine possessing cardiac type 5 adenylyl cyclase inhibitory property did not affect cardiohemodynamic or electrophysiological variables in the halothane-anesthetized dogs.

    PubMed

    Wada, Takeshi; Nakamura, Yuji; Cao, Xin; Ohara, Hiroshi; Izumi-Nakaseko, Hiroko; Ando, Kentaro; Nakazato, Yuji; Sugiyama, Atsushi

    2016-02-01

    Vidarabine has been used for the treatment of patients with local and systemic herpes virus infection; moreover, it was recently reported that it inhibits cardiac type 5 adenylyl cyclase. Furthermore, vidarabine has been shown to suppress atrial fibrillation and improve congestive heart failure in experimental models of mice induced by the isoproterenol infusion. Since information that can explain its experimentally demonstrated efficacy against congestive heart failure and atrial fibrillation remains limited, in this study we precisely assessed cardio-electropharmacological effect using the halothane-anesthetized canine model. Vidarabine was intravenously administrated in three escalating doses of 1, 10, 100 mg/kg over 10 min with a pause between the doses (n = 4). Meanwhile, the vehicle dimethyl sulfoxide in volumes of 0.033, 0.033 and 0.33 mL/kg was intravenously administrated in the same manner as was vidarabine (n = 4). No significant difference was detected in any cardiohemodynamic or electrophysiological variables between the vehicle- and vidarabine-treated groups, which indicates that effective doses of vidarabine adequately inhibiting type 5 adenylyl cyclase did not affect the cardiovascular variables in vivo at all, showing its cardiac safety profile under physiological condition. Thus, the clinical utility of vidarabine might be limited to the pathological situation including congestive heart failure with increased adrenergic tone and/or sympathetic nerve-dependent atrial fibrillation. PMID:26763398

  20. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  1. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis

    PubMed Central

    Zhang, Xuebin; Candas, Mehmet; Griko, Natalya B.; Taussig, Ronald; Bulla, Lee A.

    2006-01-01

    Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg2+-dependent cytotoxic event upon binding of the toxin to its receptor BT-R1. Here we show that binding of Cry toxin to BT-R1 provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (Gαs) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R1 in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides. PMID:16788061

  2. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis.

    PubMed

    Zhang, Xuebin; Candas, Mehmet; Griko, Natalya B; Taussig, Ronald; Bulla, Lee A

    2006-06-27

    Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg(2+)-dependent cytotoxic event upon binding of the toxin to its receptor BT-R(1). Here we show that binding of Cry toxin to BT-R(1) provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G(alphas)) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R(1) in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides.

  3. The type 3 adenylyl cyclase is required for the survival and maturation of newly generated granule cells in the olfactory bulb.

    PubMed

    Luo, Jie; Chen, Xuanmao; Pan, Yung-Wei; Lu, Song; Xia, Zhengui; Storm, Daniel R

    2015-01-01

    The type 3 adenylyl cyclase (AC3) is localized to olfactory cilia in the main olfactory epithelium (MOE) and primary cilia in the adult mouse brain. Although AC3 has been strongly implicated in odor perception and olfactory sensory neuron (OSN) targeting, its role in granule cells (GCs), the most abundant interneurons in the main olfactory bulb (MOB), remains largely unknown. Here, we report that the deletion of AC3 leads to a significant reduction in the size of the MOB as well as the level of adult neurogenesis. The cell proliferation and cell cycle in the subventricular zone (SVZ), however, are not suppressed in AC3-/- mice. Furthermore, AC3 deletion elevates the apoptosis of GCs and disrupts the maturation of newly formed GCs. Collectively, our results identify a fundamental role for AC3 in the development of adult-born GCs in the MOB.

  4. Adenylyl cyclase is required for cAMP production, growth, conidial germination, and virulence in the citrus green mold pathogen Penicillium digitatum.

    PubMed

    Wang, Weili; Wang, Mingshuang; Wang, Jiye; Zhu, Congyi; Chung, Kuang-Ren; Li, Hongye

    2016-11-01

    Penicillium digitatum is the causative agent of green mold decay on citrus fruit. The cAMP-mediated signaling pathway plays an important role in the transduction of extracellular signals and has been shown to regulate a wide range of developmental processes and pathogenicity in fungal pathogens. We cloned and characterized a Pdac1 gene of P. digitatum, which encodes a polypeptide similar to fungal adenylyl cyclases. Using a loss-of-function mutation in the Pdac1 gene we demonstrated a critical requirement for hyphal growth and conidial germination. Deletion of Pdac1 resulted in decreased accumulation of cAMP and down-regulation of genes encoding a G protein α subunit, both catalytic and regulatory subunits of PKA, and two transcriptional regulators StuA and Som1. Fungal mutants lacking Pdac1 produced abundant conidia, which failed to germinate effectively and displayed an elevated sensitivity to heat treatment. Pdac1 mutant failed to utilize carbohydrates effectively and thus displayed severe growth retardation on rich and synthetic media. Slow growth seen in the Pdac1 mutants could be due to a defect in nutrient sensing and acquisition. Quantitative RT-PCR analysis revealed that Pdac1 was primarily expressed at the early stage of infection. Fungal pathogenicity assayed on citrus fruit revealed that P. digitatum strains impaired for Pdac1 delayed lesion formation. Our results highlight important regulatory roles of adenylyl cyclase-mediated cAMP production in P. digitatum and provide insights into the critical role of cAMP in fungal growth, development and virulence.

  5. Adenylyl cyclase is required for cAMP production, growth, conidial germination, and virulence in the citrus green mold pathogen Penicillium digitatum.

    PubMed

    Wang, Weili; Wang, Mingshuang; Wang, Jiye; Zhu, Congyi; Chung, Kuang-Ren; Li, Hongye

    2016-11-01

    Penicillium digitatum is the causative agent of green mold decay on citrus fruit. The cAMP-mediated signaling pathway plays an important role in the transduction of extracellular signals and has been shown to regulate a wide range of developmental processes and pathogenicity in fungal pathogens. We cloned and characterized a Pdac1 gene of P. digitatum, which encodes a polypeptide similar to fungal adenylyl cyclases. Using a loss-of-function mutation in the Pdac1 gene we demonstrated a critical requirement for hyphal growth and conidial germination. Deletion of Pdac1 resulted in decreased accumulation of cAMP and down-regulation of genes encoding a G protein α subunit, both catalytic and regulatory subunits of PKA, and two transcriptional regulators StuA and Som1. Fungal mutants lacking Pdac1 produced abundant conidia, which failed to germinate effectively and displayed an elevated sensitivity to heat treatment. Pdac1 mutant failed to utilize carbohydrates effectively and thus displayed severe growth retardation on rich and synthetic media. Slow growth seen in the Pdac1 mutants could be due to a defect in nutrient sensing and acquisition. Quantitative RT-PCR analysis revealed that Pdac1 was primarily expressed at the early stage of infection. Fungal pathogenicity assayed on citrus fruit revealed that P. digitatum strains impaired for Pdac1 delayed lesion formation. Our results highlight important regulatory roles of adenylyl cyclase-mediated cAMP production in P. digitatum and provide insights into the critical role of cAMP in fungal growth, development and virulence. PMID:27664719

  6. Sensing Positive versus Negative Reward Signals through Adenylyl Cyclase-Coupled GPCRs in Direct and Indirect Pathway Striatal Medium Spiny Neurons

    PubMed Central

    Nair, Anu G.; Eriksson, Olivia; Vincent, Pierre

    2015-01-01

    Transient changes in striatal dopamine (DA) concentration are considered to encode a reward prediction error (RPE) in reinforcement learning tasks. Often, a phasic DA change occurs concomitantly with a dip in striatal acetylcholine (ACh), whereas other neuromodulators, such as adenosine (Adn), change slowly. There are abundant adenylyl cyclase (AC) coupled GPCRs for these neuromodulators in striatal medium spiny neurons (MSNs), which play important roles in plasticity. However, little is known about the interaction between these neuromodulators via GPCRs. The interaction between these transient neuromodulator changes and the effect on cAMP/PKA signaling via Golf- and Gi/o-coupled GPCR are studied here using quantitative kinetic modeling. The simulations suggest that, under basal conditions, cAMP/PKA signaling could be significantly inhibited in D1R+ MSNs via ACh/M4R/Gi/o and an ACh dip is required to gate a subset of D1R/Golf-dependent PKA activation. Furthermore, the interaction between ACh dip and DA peak, via D1R and M4R, is synergistic. In a similar fashion, PKA signaling in D2+ MSNs is under basal inhibition via D2R/Gi/o and a DA dip leads to a PKA increase by disinhibiting A2aR/Golf, but D2+ MSNs could also respond to the DA peak via other intracellular pathways. This study highlights the similarity between the two types of MSNs in terms of high basal AC inhibition by Gi/o and the importance of interactions between Gi/o and Golf signaling, but at the same time predicts differences between them with regard to the sign of RPE responsible for PKA activation. SIGNIFICANCE STATEMENT Dopamine transients are considered to carry reward-related signal in reinforcement learning. An increase in dopamine concentration is associated with an unexpected reward or salient stimuli, whereas a decrease is produced by omission of an expected reward. Often dopamine transients are accompanied by other neuromodulatory signals, such as acetylcholine and adenosine. We highlight the

  7. The Adenylyl Cyclase Plays a Regulatory Role in the Morphogenetic Switch from Vegetative to Pathogenic Lifestyle of Fusarium graminearum on Wheat

    PubMed Central

    Bormann, Jörg; Boenisch, Marike Johanne; Brückner, Elena; Firat, Demet; Schäfer, Wilhelm

    2014-01-01

    Cyclic 3′,5′-adenosine monophosphate (cAMP) is a nucleotide derived from adenosine triphosphate that acts as a second messenger throughout all kingdoms. Intracellular cAMP levels are synthesized by a membrane-bound protein, the adenylyl cyclase. In order to analyze the function of this gene and the importance of cAMP in the life cycle of the cereal pathogen Fusarium graminearum, the adenylyl cyclase gene (FGSG_01234) was deleted by gene replacement (ΔFgac1). The ΔFgac1 mutant displayed a drastically reduced growth on agar medium which could be rescued by a cAMP analogon. Furthermore, the ΔFgac1 mutant was unable to produce perithecia on detached wheat nodes. However, artificial conditions like carrot agar allowed perithecia development. Pathogenicity towards wheat was drastically reduced in ΔFgac1 compared to the wild type. Point-inoculated spikelets showed only small lesions but no typical head blight disease symptoms. Fluorescence microscopy using dsRed-expressing strains revealed that the ΔFgac1 strain was unable to develop any complex infection structures like lobate appressoria and infection cushions. Instead, hyphal anastomosis occurs frequently. Scanning electron microscopy demonstrated the lack of fungal penetration. Hence, the formation of compound appressoria seems to be essential for infection of wheat. Hyphae on flower leaves produced huge amounts of new conidia, thereby circumventing the infection cycle. This abundant sporulation on wheat epidermis was not observed in wild type. Intriguingly, the Fgac1 deletion mutant was able to infect maize cobs as wild type, indicating that cAMP signaling is not important for maize infection. The ΔFgac1 mutant was unable to produce the mycotoxin deoxynivalenol both in vitro and during wheat infection. In this study, we show that cAMP signaling controls important cellular processes such as development of infection structures, pathogenicity, secondary metabolite production and sexual reproduction. For the

  8. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  9. Adenylyl Cyclase Anchoring by a Kinase Anchor Protein AKAP5 (AKAP79/150) Is Important for Postsynaptic β-Adrenergic Signaling*

    PubMed Central

    Zhang, Mingxu; Patriarchi, Tommaso; Stein, Ivar S.; Qian, Hai; Matt, Lucas; Nguyen, Minh; Xiang, Yang K.; Hell, Johannes W.

    2013-01-01

    Recent evidence indicates that the A kinase anchor protein AKAP5 (AKAP79/150) interacts not only with PKA but also with various adenylyl cyclase (AC) isoforms. However, the physiological relevance of AC-AKAP5 binding is largely unexplored. We now show that postsynaptic targeting of AC by AKAP5 is important for phosphorylation of the AMPA-type glutamate receptor subunit GluA1 on Ser-845 by PKA and for synaptic plasticity. Phosphorylation of GluA1 on Ser-845 is strongly reduced (by 70%) under basal conditions in AKAP5 KO mice but not at all in D36 mice, in which the PKA binding site of AKAP5 (i.e. the C-terminal 36 residues) has been deleted without affecting AC association with GluA1. The increase in Ser-845 phosphorylation upon β-adrenergic stimulation is much more severely impaired in AKAP5 KO than in D36 mice. In parallel, long term potentiation induced by a 5-Hz/180-s tetanus, which mimics the endogenous θ-rhythm and depends on β-adrenergic stimulation, is only modestly affected in acute forebrain slices from D36 mice but completely abrogated in AKAP5 KO mice. Accordingly, anchoring of not only PKA but also AC by AKAP5 is important for regulation of postsynaptic functions and specifically AMPA receptor activity. PMID:23649627

  10. Role of rut adenylyl cyclase in the ensemble regulation of presynaptic terminal excitability: reduced synaptic strength and precision in a Drosophila memory mutant.

    PubMed

    Ueda, Atsushi; Wu, Chun-Fang

    2009-01-01

    Although modulation of presynaptic terminal excitability can profoundly affect transmission efficacy, how excitability along axonal terminal branches is regulated requires further investigations. We performed focal patch recording in Drosophila larval neuromuscular junctions (NMJs) to monitor the activity of individual synaptic boutons along the presynaptic terminal. Analysis of the learning mutant rutabaga (rut) suggests a tight regulation of presynaptic terminal excitability by rut adenylyl cyclase (AC) that is responsible for Ca2+/calmodulin-dependent cAMP synthesis. Focal excitatory junctional currents (ejcs) demonstrated that disrupted cAMP metabolism in rut mutant boutons leads to decreased transmitter release, coupled with temporal dispersion and amplitude fluctuation of ejcs during repetitive activity. Strikingly, rut motor terminals displayed greatly increased variability among corresponding terminal branches of identified NMJs in different preparations. However, boutons throughout single terminal branches were relatively uniform in either WT or rut mutant larvae. The use of electrotonic depolarization to directly evoke transmitter release from axonal terminals revealed that variability in neurotransmission originated from varying degrees of weakened excitability in rut terminals. Pharmacological treatments and axonal action potential recordings raised the possibility that defective rut AC resulted in reduced Ca2+ currents in the nerve terminal. Thus, our data indicate that rut AC not only affects transmitter release machinery, but also plays a previously unsuspected role in local excitability control, both contributing to transmission level and precision along the entire axonal terminal. PMID:19101836

  11. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants.

    PubMed

    Zhou, G L; Miyazaki, Y; Nakagawa, T; Tanaka, K; Shishido, K; Matsuda, H; Kawamukai, M

    1998-04-01

    The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function. PMID:9579081

  12. Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

    NASA Astrophysics Data System (ADS)

    Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

    2016-08-01

    Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

  13. Adenylyl cyclase anchoring by a kinase anchor protein AKAP5 (AKAP79/150) is important for postsynaptic β-adrenergic signaling.

    PubMed

    Zhang, Mingxu; Patriarchi, Tommaso; Stein, Ivar S; Qian, Hai; Matt, Lucas; Nguyen, Minh; Xiang, Yang K; Hell, Johannes W

    2013-06-14

    Recent evidence indicates that the A kinase anchor protein AKAP5 (AKAP79/150) interacts not only with PKA but also with various adenylyl cyclase (AC) isoforms. However, the physiological relevance of AC-AKAP5 binding is largely unexplored. We now show that postsynaptic targeting of AC by AKAP5 is important for phosphorylation of the AMPA-type glutamate receptor subunit GluA1 on Ser-845 by PKA and for synaptic plasticity. Phosphorylation of GluA1 on Ser-845 is strongly reduced (by 70%) under basal conditions in AKAP5 KO mice but not at all in D36 mice, in which the PKA binding site of AKAP5 (i.e. the C-terminal 36 residues) has been deleted without affecting AC association with GluA1. The increase in Ser-845 phosphorylation upon β-adrenergic stimulation is much more severely impaired in AKAP5 KO than in D36 mice. In parallel, long term potentiation induced by a 5-Hz/180-s tetanus, which mimics the endogenous θ-rhythm and depends on β-adrenergic stimulation, is only modestly affected in acute forebrain slices from D36 mice but completely abrogated in AKAP5 KO mice. Accordingly, anchoring of not only PKA but also AC by AKAP5 is important for regulation of postsynaptic functions and specifically AMPA receptor activity.

  14. Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression.

    PubMed

    Verkerke-van Wijk, I; Fukuzawa, M; Devreotes, P N; Schaap, P

    2001-06-01

    cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, which is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA.

  15. High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.

    PubMed

    Blum, Ailisa; Benfield, Aurélie H; Stiller, Jiri; Kazan, Kemal; Batley, Jacqueline; Gardiner, Donald M

    2016-05-01

    Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes. PMID:26932301

  16. Acutely administered melatonin decreases somatostatin-binding sites and the inhibitory effect of somatostatin on adenylyl cyclase activity in the rat hippocampus.

    PubMed

    Izquierdo-Claros, Rosa María; Boyano-Adánez Md, María del Carmen; Arilla-Ferreiro, Eduardo

    2004-03-01

    Melatonin is known to increase neuronal activity in the hippocampus, an effect contrary to that of somatostatin (somatotropin release-inhibiting factor, SRIF). Thus, the aim of this study was to investigate whether the somatostatinergic system is implicated in the mechanism of action of melatonin in the rat hippocampus. One group of rats was injected a single dose of melatonin [25 microg/kg subcutaneously (s.c.)] or saline containing ethanol (0.5%, s.c.) and killed 5 hr later. Melatonin significantly decreased the SRIF-like immunoreactivity levels and induced a significant decrease in the density of SRIF receptors as well as in the dissociation constant (Kd). SRIF-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase activity was markedly decreased in hippocampal membranes from melatonin-treated rats. The functional activity of Gi proteins was similar in hippocampal membranes from melatonin-treated and control rats. Western blot analyses revealed that melatonin administration did not alter Gialpha1 or Gialpha2 levels. To determine if the changes observed were related to melatonin-induced activation of central melatonin receptors, a melatonin receptor antagonist, luzindole, was administered prior to melatonin injection. Pretreatment with luzindole (10 mg/kg, s.c.) did not alter the melatonin-induced effects on the above-mentioned parameters and luzindole, alone, had no observable effect. The present results demonstrate that melatonin decreases the activity of the SRIF receptor-effector system in the rat hippocampus, an effect which is apparently not mediated by melatonin receptors. As SRIF exerts an opposite effect to that of melatonin on hippocampal neuronal activity, it is possible that the SRIFergic system could be implicated in the mechanism of action of melatonin in the rat.

  17. Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors of Bordetella pertussis Adenylyl Cyclase Toxin

    PubMed Central

    Geduhn, Jens; Dove, Stefan; Shen, Yuequan; Tang, Wei-Jen; König, Burkhard

    2011-01-01

    Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important for reducing potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Pharmacol 72:526–535, 2007). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2, and 5. Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥100-fold higher than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT nucleotides facilitate development of a high-throughput screening assay. PMID:20962032

  18. Molecular cloning of growth hormone-releasing hormone/pituitary adenylyl cyclase-activating polypeptide in the frog Xenopus laevis: brain distribution and regulation after castration.

    PubMed

    Hu, Z; Lelievre, V; Tam, J; Cheng, J W; Fuenzalida, G; Zhou, X; Waschek, J A

    2000-09-01

    Pituitary adenylyl cyclase-activating peptide (PACAP) appears to regulate several neuroendocrine functions in the frog, but its messenger RNA (mRNA) structure and brain distribution are unknown. To understand the potential role of PACAP in the male frog hypothalamic-pituitary-gonadal axis, we cloned the frog Xenopus laevis PACAP mRNA and determined its distribution in the brain. We then analyzed the castration-induced alterations of mRNA expression for PACAP and its selective type I receptor (PAC1) in the hypothalamic anterior preoptic area, a region known to regulate reproductive function. The PACAP mRNA encodes a peptide precursor predicted to give rise to both GH-releasing hormone and PACAP. The deduced peptide sequence of PACAP-38 was nearly identical to that of human PACAP with one amino acid substitution. Abundant PACAP mRNA was detected in the brain, but not several other tissues, including the testis. In situ hybridization revealed strong expression of the PACAP gene in the dorsal pallium, ventral hypothalamus, and nuclei of cerebellum. PACAP mRNA signals were weak to moderate in the hypothalamic anterior preoptic area and were absent in the pituitary. Castration induced an increase in the expression of PACAP and PAC1 receptor mRNAs in the hypothalamic anterior preoptic area after 3 days. Replacement with testosterone prevented the castration-induced changes. These results provide a molecular basis for studying the physiological functions of PACAP in frog brain and suggest that PACAP may be involved in the feedback regulation of hypothalamic-pituitary-gonadal axis.

  19. Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts.

    PubMed

    Ostrom, Rennolds S; Naugle, Jennifer E; Hase, Miki; Gregorian, Caroline; Swaney, James S; Insel, Paul A; Brunton, Laurence L; Meszaros, J Gary

    2003-07-01

    Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts. PMID:12711600

  20. [Structural and functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in the muscle of a worm Lumbricus terrestris].

    PubMed

    Shpakov, A O; Shpakova, E A; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2008-01-01

    It has been shown for the first time that biogenic amines (catecholamines and tryptophane derivatives) stimulate dose-dependently activity of adenylyl cyclase (AC) and GTP-binding of G-proteins in muscle of the cutaneous-muscle bag of the earthworm Lumbricus terrestris. By efficiency of their stimulating action on the AC activity, biogenic amines can be arranged in the following sequence: octopamine > tyramine > tryptamine = serotonin > dopamine > isoproterenol = adrenalin. The sequence of efficiency of their action on GTP-binding is somewhat different: serotonin > tryptamine > octopamine > dopamine = tyramine > adrenaline > isoproterenol. Sensitivity of AC and G-proteins in the worm muscle to biogenic amines is similar with that in smooth muscle of the molluse Anodonta cygnea (invertebrates), but differs markedly by this parameter from the rat myocardium (vertebrates). It has also been revealed that AC in the worm muscle is regulated by peptide hormones relaxin and somatostatin whose action is comparable with that in the mollusk muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of C-terminal peptides of alpha-subunits of G-proteins of the stimulatory (385-394 Galpha(s)) and inhibitory (346-355 Galpha(i2)) types that disrupt selectively the hormonal signal transduction realized via G(s)- and G(i)-proteins, respectively, allowed establishing that the AC-stimulating effects of relaxin, octopamine, tyramine, and dopamine in the worm muscle are realized via the receptors coupled functionally with G(s)-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with G(i)-protein, whereas serotonin and tryptamine activate both types of G-proteins. PMID:18959208

  1. Circadian rhythm of contrast sensitivity is regulated by a dopamine-neuronal PAS-domain protein 2-adenylyl cyclase 1 signaling pathway in retinal ganglion cells.

    PubMed

    Hwang, Christopher K; Chaurasia, Shyam S; Jackson, Chad R; Chan, Guy C-K; Storm, Daniel R; Iuvone, P Michael

    2013-09-18

    Spatial variation in light intensity, called spatial contrast, comprises much of the visual information perceived by mammals, and the relative ability to detect contrast is referred to as contrast sensitivity (Purves et al., 2012). Recently, retinal dopamine D4 receptors (D4Rs) have been implicated in modulating contrast sensitivity (Jackson et al., 2012); however, the cellular and molecular mechanisms have not been elucidated. Our study demonstrates a circadian rhythm of contrast sensitivity that peaks during the daytime, and that its regulation involves interactions of D4Rs, the clock gene Npas2, and the clock-controlled gene adenylyl cyclase 1 (Adcy1) in a subset of retinal ganglion cells (RGCs). Targeted disruption of the gene encoding D4Rs reduces the amplitude of the contrast sensitivity rhythm by reducing daytime sensitivity and abolishes the rhythmic expression of Npas2 and Adcy1 mRNA in the ganglion cell layer (GCL) of the retina. Npas2(-/-) and Adcy1(-/-) mice show strikingly similar reductions in the contrast sensitivity rhythm to that in mice lacking D4Rs. Moreover, Adcy1 transcript rhythms were abolished in the GCL of Npas2(-/-) mice. Luciferase reporter assays demonstrated that the Adcy1 promoter is selectively activated by neuronal PAS-domain protein 2 (NPAS2)/BMAL1. Our results indicate that the contrast sensitivity rhythm is modulated by D4Rs via a signaling pathway that involves NPAS2-mediated circadian regulation of Adcy1. Hence, we have identified a circadian clock mechanism in a subset of RGCs that modulates an important aspect of retinal physiology and visual processing.

  2. Differential expression of gamma-aminobutyric acid type B receptor subunit mRNAs in the developing nervous system and receptor coupling to adenylyl cyclase in embryonic neurons.

    PubMed

    Martin, Stella C; Steiger, Janine L; Gravielle, María Clara; Lyons, Helen R; Russek, Shelley J; Farb, David H

    2004-05-17

    gamma-Aminobutyric acid type B receptors (GABA(B)Rs) mediate both slow inhibitory synaptic activity in the adult nervous system and motility signals for migrating embryonic cortical cells. Previous papers have described the expression of GABA(B)Rs in the adult brain, but the expression and functional significance of these gene products in the embryo are largely unknown. Here we examine GABA(B)R expression from rat embryonic day 10 (E10) to E18 compared with adult and ask whether embryonic cortical neurons contain functional GABA(B)R. GABA(B)R1 transcript levels greatly exceed GABA(B)R2 levels in the developing neural tube at E11, and olfactory bulb and striatum at E17 but equalize in most regions of adult nervous tissue, except for the glomerular and granule cell layers of the main olfactory bulb and the striatum. Consistent with expression differences, the binding affinity of GABA for GABA(B)Rs is significantly lower in adult striatum compared with cerebellum. Multiple lines of evidence from in situ hybridization, RNase protection, and real-time PCR demonstrate that GABA(B)R1a, GABA(B)R1b, GABA(B)R1h (a subunit subtype, lacking a sushi domain, that we have identified in embryonic rat brain), GABA(B)R2, and GABA(B)L transcript levels are not coordinately regulated. Despite the functional requirement for a heterodimer of GABA(B)R subunits, the expression of each subunit mRNA is under independent control during embryonic development, and, by E18, GABA(B)Rs are negatively coupled to adenylyl cyclase in neocortical neurons. The presence of embryonic GABA(B)R transcripts and protein and functional receptor coupling indicates potentially important roles for GABA(B)Rs in modulation of synaptic transmission in the developing embryonic nervous system.

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

    PubMed Central

    Lila, T; Drubin, D G

    1997-01-01

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

  4. Lack of an effect of collecting duct-specific deletion of adenylyl cyclase 3 on renal Na+ and water excretion or arterial pressure

    PubMed Central

    Kittikulsuth, Wararat; Stuart, Deborah; Van Hoek, Alfred N.; Stockand, James D.; Bugaj, Vladislav; Mironova, Elena; Blount, Mitsi A.

    2014-01-01

    cAMP is a key mediator of connecting tubule and collecting duct (CD) Na+ and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na+ or K+ excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na+ channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na+ and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na+ and water excretion in general or vasopressin regulation of CD function in particular. PMID:24431204

  5. Gi/o-Coupled Receptors Compete for Signaling to Adenylyl Cyclase in SH-SY5Y Cells and Reduce Opioid-Mediated cAMP Overshoot

    PubMed Central

    Levitt, Erica S.; Purington, Lauren C.

    2011-01-01

    Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the μ-opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to δ-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), α2-adrenergic (α2AR), cannabinoid 1, and serotonin 1A receptors. We further demonstrate that all receptor pairs share a common pool of AC. The MOR agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and α2AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal. PMID:21098043

  6. Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts.

    PubMed

    Ostrom, Rennolds S; Naugle, Jennifer E; Hase, Miki; Gregorian, Caroline; Swaney, James S; Insel, Paul A; Brunton, Laurence L; Meszaros, J Gary

    2003-07-01

    Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.

  7. Dysregulation of TrkB phosphorylation and proBDNF protein in adenylyl cyclase 1 and 8 knockout mice in a model of fetal alcohol spectrum disorder.

    PubMed

    Susick, Laura L; Chrumka, Alexandria C; Hool, Steven M; Conti, Alana C

    2016-03-01

    Brain-derived neurotrophic factor (BDNF) mediates neuron growth and is regulated by adenylyl cyclases (ACs). Mice lacking AC1/8 (DKO) have a basal reduction in the dendritic complexity of medium spiny neurons in the caudate putamen and demonstrate increased neurotoxicity in the striatum following acute neonatal ethanol exposure compared to wild type (WT) controls, suggesting a compromise in BDNF regulation under varying conditions. Although neonatal ethanol exposure can negatively impact BDNF expression, little is known about the effect on BDNF receptor activation and its downstream signaling, including Akt activation, an established neuroprotective pathway. Therefore, here we determined the effects of AC1/8 deletion and neonatal ethanol administration on BDNF and proBDNF protein expression, and activation of tropomyosin-related kinase B (TrkB), Akt, ERK1/2, and PLCγ. WT and DKO mice were treated with a single dose of 2.5 g/kg ethanol or saline at postnatal days 5-7 to model late-gestational alcohol exposure. Striatal and cortical tissues were analyzed using a BDNF enzyme-linked immunosorbent assay or immunoblotting for proBDNF, phosphorylated and total TrkB, Akt, ERK1/2, and PLCɣ1. Neither postnatal ethanol exposure nor AC1/8 deletion affected total BDNF protein expression at any time point in either region examined. Neonatal ethanol increased the expression of proBDNF protein in the striatum of WT mice 6, 24, and 48 h after exposure, with DKO mice demonstrating a reduction in proBDNF expression 6 h after exposure. Six and 24 h after ethanol administration, phosphorylation of full-length TrkB in the striatum was significantly reduced in WT mice, but was significantly increased in DKO mice only at 24 h. Interestingly, 48 h after ethanol, both WT and DKO mice demonstrated a reduction in phosphorylated full-length TrkB. In addition, Akt and PLCɣ1 phosphorylation was also decreased in ethanol-treated DKO mice 48 h after injection. These data demonstrate

  8. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains.

    PubMed

    Ostrom, Rennolds S; Liu, Xiaoqiu; Head, Brian P; Gregorian, Caroline; Seasholtz, Tammy M; Insel, Paul A

    2002-11-01

    A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the

  9. [Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

    PubMed

    Shpakov, A O; Shpakova, E A; Tarasenko, I I; Derkach, K V

    2014-01-01

    The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide

  10. [Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system].

    PubMed

    Shpakov, A O; Gur'ianov, I A; Kuznetsova, L A; Plesneva, S A; Zakharova, E T; Vlasov, G P; Pertseva, M N

    2006-01-01

    The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.

  11. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains.

    PubMed

    Ostrom, Rennolds S; Liu, Xiaoqiu; Head, Brian P; Gregorian, Caroline; Seasholtz, Tammy M; Insel, Paul A

    2002-11-01

    A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the

  12. [Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

    PubMed

    Shpakov, A O; Shpakova, E A; Tarasenko, I I; Derkach, K V

    2014-01-01

    The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide

  13. Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by beta-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex.

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

    We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

  14. The vasorelaxant effect of 8(17),12E,14-labdatrien-18-oic acid involves stimulation of adenylyl cyclase and cAMP/PKA pathway: Evidences by pharmacological and molecular docking studies.

    PubMed

    Ribeiro, Luciano A A; Alencar Filho, Edilson B; Coelho, Maisa C; Silva, Bagnólia A

    2015-10-01

    The relaxant effect of 8(17),12E,14-labdatrien-18-oic acid (LBD) was investigated on isolated aortic rings and compared with forskolin (FSK), a standard and potent activator of adenylyl cyclase (AC) with relaxing effect. The presence of potassium channel blockers, such as glibenclamide (ATP-blocker), apamin (SKCa-blocker), charybdotoxin (BKCa-blocker) did not significantly affect either the LBD or FSK concentration-response curves. However, in the presence of 4-aminopyridine (KV-blocker), the relaxant effect for both diterpenes was significantly attenuated, with reduction of its relative potencies. Moreover, the relaxation induced by 8-Br-cAMP, an analog of cAMP, was also significantly attenuated in the same conditions, i.e., in the presence of 4-aminopyridine. The presence of aminophylline, a nonselective phosphodiesterase inhibitor, caused a significant increasing in the potency for both LBD and FSK. On the other hand, the presence of Rp-cAMPS, a selective PKA-inhibitor, significantly attenuated the relaxant effect of LBD. In this work, in the same experimental conditions, both labdane-type diterpenes presented remarkably similar results; FSK, however, presented a higher potency (100-fold) than LBD. Thus, the hypothesis that LBD could be a novel AC-activator emerged. To assess that hypothesis, computational molecular docking studies were performed. Crystallographic structure of adenylyl cyclase/forskolin complex (1AB8) was obtained from RSCB Protein Data Bank and used to compare the modes of interaction of the native ligand and LBD. The computational data shows many similarities between LBD and FSK concerning the interaction with the regulatory site of AC. Taken together, the results presented here pointed to LBD as a novel AC-activator. PMID:26144373

  15. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT1A receptor-adenylyl cyclase axis

    PubMed Central

    Stewart, Adele; Maity, Biswanath; Wunsch, Amanda M.; Meng, Fantao; Wu, Qi; Wemmie, John A.; Fisher, Rory A.

    2014-01-01

    Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT1ARs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT1AR-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT1AR-expressing cells implicated in mood disorders. RGS6−/− mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT1AR blockade. Effects of the SSRI fluvoxamine and 5-HT1AR agonist 8-OH-DPAT were also potentiated in RGS6+/− mice. The phenotype of RGS6−/− mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gαi-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6−/− animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents.—Stewart, A., Maity, B., Wunsch, A. M., Meng, F., Wu, Q., Wemmie, J. A., Fisher, R. A. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT1A receptor-adenylyl cyclase axis. PMID:24421401

  16. [THE EFFECTS OF LONG-TERM METFORMIN TREATMENT ON THE ACTIVITY OF ADENYLYL CYCLASE SYSTEM AND NO-SYNTHASES IN THE BRAIN AND THE MYOCARDIUM OF RATS WITH OBESITY].

    PubMed

    Derkach, K V; Kuznetsova, L A; Sharova, T S; Ignatieva, P A; Bondareva, V M; Shpakov, A O

    2015-01-01

    Biguanide metformin, which is widely used for the treatment of type 2 diabetes mellitus, improves carbohydrate and lipid metabolism and shows a pronounced cardio- and neuroprotective effects. It is assumed that an important role in these effects of metformin plays its ability to positively influence the activity of NO-synthase catalyzing the synthesis of NO, the most important vasodilator, and the activity of hormone-sensitive adenylyl cyclase signaling system (ACSS. To prove this, we have carried out a study whose purpose was to study the effect of long-term metformin treatment on the metabolic rates in obese rats, as well as on the activity of ACSS and NO-synthase in the myocardium and the brain of these animals. The metformin treatment of Wistar rats with obesity induced by high-fat diet was carried out for 2 months (daily dose of 200 mg/kg). The treatment with metformin led to a decrease in body weight and body fat, reduced glucose and insulin levels as well as reduced insulin resistance index HOMA-IR, improved glucose tolerance, and decreased the level of atherogenic forms of cholesterol. In the myocardium of obese rats, the attenuation of ACSS stimulation induced by the agonists of β1/β2-adrenergic receptors (AR) and the strengthening of β3-AR signaling has been found. At the same time, in the myocardium of animals treated with metformin, the regulation of ACSS by adrenergic agonists was restored, and the ratio of β-AR-signaling pathways returned to normal. In the brain of rats treated with metformin, adenylyl cyclase stimulating effects of serotonin and agonists of type 4 melanocortin receptors, which had been weakenend for obesity, were restored. Metformin treatment completely restored activity of total and endothelial NO-synthase in the myocardium decreased in obesity. It as also shown that metformin treatment induced hyperactivation of NO-synthase in the myocardium and brain of healthy animals. Thus, we conclude that the effects of metformin

  17. [THE EFFECTS OF LONG-TERM METFORMIN TREATMENT ON THE ACTIVITY OF ADENYLYL CYCLASE SYSTEM AND NO-SYNTHASES IN THE BRAIN AND THE MYOCARDIUM OF RATS WITH OBESITY].

    PubMed

    Derkach, K V; Kuznetsova, L A; Sharova, T S; Ignatieva, P A; Bondareva, V M; Shpakov, A O

    2015-01-01

    Biguanide metformin, which is widely used for the treatment of type 2 diabetes mellitus, improves carbohydrate and lipid metabolism and shows a pronounced cardio- and neuroprotective effects. It is assumed that an important role in these effects of metformin plays its ability to positively influence the activity of NO-synthase catalyzing the synthesis of NO, the most important vasodilator, and the activity of hormone-sensitive adenylyl cyclase signaling system (ACSS. To prove this, we have carried out a study whose purpose was to study the effect of long-term metformin treatment on the metabolic rates in obese rats, as well as on the activity of ACSS and NO-synthase in the myocardium and the brain of these animals. The metformin treatment of Wistar rats with obesity induced by high-fat diet was carried out for 2 months (daily dose of 200 mg/kg). The treatment with metformin led to a decrease in body weight and body fat, reduced glucose and insulin levels as well as reduced insulin resistance index HOMA-IR, improved glucose tolerance, and decreased the level of atherogenic forms of cholesterol. In the myocardium of obese rats, the attenuation of ACSS stimulation induced by the agonists of β1/β2-adrenergic receptors (AR) and the strengthening of β3-AR signaling has been found. At the same time, in the myocardium of animals treated with metformin, the regulation of ACSS by adrenergic agonists was restored, and the ratio of β-AR-signaling pathways returned to normal. In the brain of rats treated with metformin, adenylyl cyclase stimulating effects of serotonin and agonists of type 4 melanocortin receptors, which had been weakenend for obesity, were restored. Metformin treatment completely restored activity of total and endothelial NO-synthase in the myocardium decreased in obesity. It as also shown that metformin treatment induced hyperactivation of NO-synthase in the myocardium and brain of healthy animals. Thus, we conclude that the effects of metformin

  18. [The influence of two-month treatment with bromocryptine on activity of the adenylyl cyclase signaling system in the myocardium and testes of rats with type 2 diabetes mellitus].

    PubMed

    Derkach, K V; Bondareva, V M; Moyseyuk, I V; Shpakov, A O

    2014-01-01

    One of the common complications of type 2 diabetes mellitus (DM2) are cardiovascular diseases and dysfunctions of the reproductive system, indicating the urgency of developing new approaches to their correction. Last years for the treatment of DM2 began to use bromocryptine (BC), the agonist of type 2 dopamine receptors, which not only restores the energy metabolism, but also prevents the development of cardiovascular diseases. However, the mechanisms and targets of BC action are poorly understood. The purpose of this study was to investigate the effect of BC treatment on functional activity of adenylyl cyclase signaling system (ACSS) in the myocardium and testes of male rats with DM2, which is caused by high-fat diet and treatment with streptozotocin (25 mg/kg). The treatment with BC (60 days, orally at a dose of 0.6 mg/kg once every two days) was started 90 days after the beginning of high-fat diet. Diabetic rats had an increased body weight, elevated triglycerides level, impaired glucose tolerance, and insulin resistance. The treatment with BC resulted in the restoration of glycometabolic indicators and in the improvement of insulin sensitivity. Adenylyl cyclase (AC) stimulating effects of guanylylimidodiphosphate (GppNHp), relaxin, and agonists of β-adrenergic receptors (β3-AR)--isoproterenol and norepinephrine were decreased in the miocardium of the diabetic rats. The corresponding effects of the β-agonists BRL-37344 and CL-316243 was preserved. The inhibitory effect of somatostatin on forskolin-stimulated AC activity was attenuated, while the inhibitory effect of noradrenaline mediated through α2-AR increased. The treatment with BC resulted in the normalization of the adrenergic signaling in the myocardium and partially restoration of AC effects of relaxin and somatostatin. In the testes of diabetic rats, the basal and stimulated by GppNHp, forskolin, human chorionic gonadotropin and pituitary AC-activating polypeptide AC activity were decreased, and the

  19. Cyclic nucleotide–gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee

    PubMed Central

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO–cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca2+/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO–cGMP and cAMP–PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors. PMID:24741108

  20. Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation.

    PubMed

    Bayewitch, M L; Nevo, I; Avidor-Reiss, T; Levy, R; Simonds, W F; Vogel, Z

    2000-04-01

    Prolonged G(i/o) protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G(i) protein and the generation of AC superactivation.

  1. Pituitary adenylyl cyclase activating peptide (PACAP) in the bed nucleus of the stria terminalis (BNST) increases corticosterone in male and female rats

    PubMed Central

    Lezak, K. R.; Roelke, E.; Harris, O.; Choi, I.; Edwards, S.; Gick, N.; Cocchiaro, G.; Missig, G.; Roman, C. W.; Braas, K. M.; Toufexis, D.J.; May, V.; Hammack, S. E.

    2014-01-01

    Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylate cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with several psychiatric disorders whose etiology has been associated with stressor exposure and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In rats, exposure to repeated variate stress has been shown to increase PACAP and its cognate PAC1 receptor expression in the bed nucleus of the stria terminalis (BNST), a brain region implicated in anxiety and depression-related behaviors as well as the regulation of HPA axis activity. We have argued that changes in BNST PACAP signaling may mediate the changes in emotional behavior and dysregulation of the HPA axis associated with anxiety and mood disorders. The current set of studies was designed to determine whether BNST PACAP infusion leads to activation of the HPA axis as determined by increases in plasma corticosterone. We observed an increase in plasma corticosterone levels 30 minutes following BNST PACAP38 infusion in male and female rats, which was independent of estradiol (E2) treatment in females, and we found that plasma corticosterone levels were increased at both 30 minutes and 60 minutes, but returned to baseline levels 4 hours following the highest dose. PACAP38 infusion into the lateral ventricles immediately above the BNST did not alter plasma corticosterone level, and the increased plasma corticosterone following BNST PACAP was not blocked by BNST corticotropin releasing hormone (CRH) receptor antagonism. These results support others suggesting that BNST PACAP plays a key role in regulating stress responses. PMID:24845172

  2. Ser⁄ Thr residues at α3⁄β5 loop of Gαs are important in morphine-induced adenylyl cyclase sensitization but not mitogen-activated protein kinase phosphorylation

    PubMed Central

    Seyedabadi, Mohammad; Ostad, Seyed Nasser; Albert, Paul R.; Dehpour, Ahmad R.; Rahimian, Reza; Ghazi-Khansari, Mahmoud; Ghahremani, Mohammad H.

    2015-01-01

    The signaling switch of β2-adrenergic and μ1-opioid receptors from stimulatory G-protein (Gαs) to inhibitory G-protein (Gαi) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1 ⁄ 2 activation. Post-translational modifications, including dephosphorylation of Gαs, enhance opioid receptor coupling to Gαs. In the present study, we substituted the Ser ⁄ Thr residues of Gαs at the α3 ⁄ β5 and α4 ⁄ β6 loops aiming to study the role of Gαs lacking Ser ⁄ Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC50 = 22.8 ± 3.4 μM) in Gαs-transfected S49 cyc– cells but not in nontransfected cells. However, there was no significant difference between the Gαs-wild-type (wt) and mutants. Morphine (10 μM) inhibited AC activity more efficiently in cyc– compared to Gαs-wt introduced cells (P < 0.05); however, we did not find a notable difference between Gαs-wt and mutants. Interestingly, Gαs-wt transfected cells showed more sensitization with respect to AC after chronic morphine compared to nontransfected cells (101 ± 12% versus 34 ± 6%; P < 0.001); μ1-opioid receptor interacted with Gαs, and both co-immunoprecipitated after chronic morphine exposure. Furthermore, mutation of T270A and S272A (P < 0.01), as well as T270A, S272A and S261A (P < 0.05), in α3 ⁄ β5, resulted in a higher level of AC supersensitization. ERK1⁄ 2 phosphorylation was rapidly induced by isoproterenol (by 9.5 ± 2.4-fold) and morphine (22 ± 2.2-fold) in Gαs-transfected cells; mutations of α3 ⁄ β5 and α4 ⁄ β6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that Gαs interacts with the μ1-opioid receptor, and the Ser ⁄ Thr mutation to Ala at the α3 ⁄ β5 loop of Gαs enhances morphine-induced AC sensitization. In addition, Gαs was required for

  3. Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

    PubMed

    McCue, L A; McDonough, K A; Lawrence, C E

    2000-02-01

    We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species. PMID:10673278

  4. Bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

  5. [Endolymphatic sac adenocarcinoma: case report].

    PubMed

    Silveira, Roberto Leal; Gusmão, Sebastião Silva; Pittella, José Eymard H; Santos, Sinval Pereira

    2002-09-01

    A case of endolymphatic sac adenocarcinoma is reported and the literature is reviewed. The clinical picture was presented by vertigo and progressive hearing loss caused by a tumor of the endolymphatic sac. The surgical removal was complete, via a retro and translabyrinthine approach. Endolymphatic sac tumors are locally invasive, involve the petrous bone and the mastoid. The radical surgery presents good outcome.

  6. Stimulation of Hippocampal Adenylyl Cyclase Activity Dissociates Memory Consolidation Processes for Response and Place Learning

    ERIC Educational Resources Information Center

    Martel, Guillaume; Millard, Annabelle; Jaffard, Robert; Guillou, Jean-Louis

    2006-01-01

    Procedural and declarative memory systems are postulated to interact in either a synergistic or a competitive manner, and memory consolidation appears to be a highly critical stage for this process. However, the precise cellular mechanisms subserving these interactions remain unknown. To investigate this issue, 24-h retention performances were…

  7. Cytidylyl- and Uridylyl Cyclase Activity of Bacillus anthracis Edema Factor and Bordetella pertussis CyaA

    PubMed Central

    Göttle, Martin; Dove, Stefan; Kees, Frieder; Schlossmann, Jens; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2010-01-01

    Cyclic adenosine 3′:5′-monophosphate (cAMP) and cyclic guanosine 3′:5′-monophosphate (cGMP) are second messengers for a numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3′:5′-monophosphate (cCMP) is discussed controversially, and almost nothing is known about cyclic uridine 3′:5′-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analog inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC- and UC activity was determined by a radiometric assay, using [α-32P]CTP and [α-32P]UTP as substrates, respectively, and by an HPLC method. The identity of cNMPs was confirmed by mass spectrometry. Based on available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate-specificity and exhibit cytidylyl- and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions. PMID:20521845

  8. Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability*

    PubMed Central

    Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

    2012-01-01

    Exotoxin Y (ExoY) is a type III secretion system effector found in ∼ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

  9. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S.

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  10. Haemangiopericytoma of the lacrimal sac.

    PubMed

    Watanabe, Akihide; Wu, Albert; Sun, Michelle T; Inatani, Masaru; Katori, Nobutada; Selva, Dinesh

    2016-08-01

    Haemangiopericytomas (HPCs) are rare tumours which infrequently occur in the lacrimal sac. Only 8 cases of lacrimal sac HPC have previously been reported. The authors report 2 additional cases presenting clinically with epiphora and a mass. One case recurred 3 times during an 18-year period. The other case did not recur during 51 months of follow-up. The tumours showed immunohistochemical features consistent with a diagnosis of HPC. The authors recommend wide excision for these tumours and careful long-term follow-up to detect recurrence which is not uncommon. PMID:27322416

  11. Cul-de-Sac Kids

    ERIC Educational Resources Information Center

    Hochschild, Thomas R., Jr.

    2013-01-01

    Previous research indicates that adults who live on cul-de-sac streets are more likely to have positive experiences with neighbors than residents of other street types (Brown and Werner, 1985; Hochschild Jr, 2011; Mayo Jr, 1979; Willmott, 1963). The present research ascertains whether street design has an impact on children's neighborhood…

  12. Exploring adenylylation and phosphocholination as post-translational modifications.

    PubMed

    Müller, Matthias P; Albers, Michael F; Itzen, Aymelt; Hedberg, Christian

    2014-01-01

    Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.

  13. Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

    PubMed Central

    Roelofs, J; Snippe, H; Kleineidam, R G; Van Haastert, P J

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase. PMID:11237875

  14. Digitonin effects on photoreceptor adenylate cyclase.

    PubMed

    Bitensky, M W; Gorman, R E; Miller, W H

    1972-03-24

    Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

  15. Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade.

    PubMed Central

    Varón-Castellanos, R; Havsteen, B H; García-Moreno, M; Valero-Ruiz, E; Molina-Alarcón, M; García-Cánovas, F

    1993-01-01

    A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived. PMID:8104399

  16. Beneficial Effects of Adenylyl Cyclase Type 6 (AC6) Expression Persist Using a Catalytically Inactive AC6 Mutant

    PubMed Central

    Tang, Tong; Lai, Ngai Chin; Miyanohara, Atsushi; Guo, Tracy; Tang, Rouying; Firth, Amy L.; Yuan, Jason X.; Hammond, H. Kirk

    2011-01-01

    Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation. PMID:21127130

  17. Adenylate cyclase 3: a new target for anti-obesity drug development.

    PubMed

    Wu, L; Shen, C; Seed Ahmed, M; Östenson, C-G; Gu, H F

    2016-09-01

    Obesity has become epidemic worldwide, and abdominal obesity has a negative impact on health. Current treatment options on obesity, however, still remain limited. It is then of importance to find a new target for anti-obesity drug development based upon recent molecular studies in obesity. Adenylate cyclase 3 (ADCY3) is the third member of adenylyl cyclase family and catalyses the synthesis of cAMP from ATP. Genetic studies with candidate gene and genome-wide association study approaches have demonstrated that ADCY3 genetic polymorphisms are associated with obesity in European and Chinese populations. Epigenetic studies have indicated that increased DNA methylation levels in the ADCY3 gene are involved in the pathogenesis of obesity. Furthermore, biological analyses with animal models have implicated that ADCY3 dysfunction resulted in increased body weight and fat mass, while reduction of body weight is partially explained by ADCY3 activation. In this review, we describe genomic and biological features of ADCY3, summarize genetic and epigenetic association studies of the ADCY3 gene with obesity and discuss dysfunction and activation of ADCY3. Based upon all data, we suggest that ADCY3 is a new target for anti-obesity drug development. Further investigation on the effectiveness of ADCY3 activator and its delivery approach to treat abdominal obesity has been taken into our consideration. PMID:27256589

  18. SACS: Spitzer Archival Cluster Survey

    NASA Astrophysics Data System (ADS)

    Stern, Daniel

    Emerging from the cosmic web, galaxy clusters are the most massive gravitationally bound structures in the universe. Thought to have begun their assembly at z > 2, clusters provide insights into the growth of large-scale structure as well as the physics that drives galaxy evolution. Understanding how and when the most massive galaxies assemble their stellar mass, stop forming stars, and acquire their observed morphologies in these environments remain outstanding questions. The redshift range 1.3 < z < 2 is a key epoch in this respect: elliptical galaxies start to become the dominant population in cluster cores, and star formation in spiral galaxies is being quenched. Until recently, however, this redshift range was essentially unreachable with available instrumentation, with clusters at these redshifts exceedingly challenging to identify from either ground-based optical/nearinfrared imaging or from X-ray surveys. Mid-infrared (MIR) imaging with the IRAC camera on board of the Spitzer Space Telescope has changed the landscape. High-redshift clusters are easily identified in the MIR due to a combination of the unique colors of distant galaxies and a negative k-correction in the 3-5 μm range which makes such galaxies bright. Even 90-sec observations with Spitzer/IRAC, a depth which essentially all extragalactic observations in the archive achieve, is sufficient to robustly detect overdensities of L* galaxies out to z~2. Here we request funding to embark on a ambitious scientific program, the “SACS: Spitzer Archival Cluster Survey”, a comprehensive search for the most distant galaxy clusters in all Spitzer/IRAC extragalactic pointings available in the archive. With the SACS we aim to discover ~2000 of 1.3 < z < 2.5 clusters, thus provide the ultimate catalog for high-redshift MIR selected clusters: a lasting legacy for Spitzer. The study we propose will increase by more than a factor of 10 the number of high-redshift clusters discovered by all previous surveys

  19. SAC Availability for the IRIS Community

    SciTech Connect

    Goldstein, P; Snoke, A

    2005-04-06

    SAC (also known as SAC2000) is a signal processing and analysis code that has been developed by Lawrence Livermore National Laboratory (LLNL) over the past 20+ years for a variety of seismic and geophysical research projects. SAC has evolved into a general purpose interactive program designed for the study of sequential signals, especially time-series data. Emphasis has been placed on analysis tools used by research seismologists in the detailed study of seismic events. Analysis capabilities include general arithmetic operations, Fourier transforms, three spectral estimation techniques, IIR and FIR filtering, signal stacking, decimation, interpolation, correlation, and seismic phase picking. SAC also contains an extensive graphics capability. SAC is used extensively by the seismic community because: (1) it has a broad range of well-tested, efficient data analysis capabilities (examples include: data inspection, phase picking, signal correction, quality control, unary and binary data operations, travel-time analysis, spectral analysis including high-resolution spectral estimation, spectrograms and binary sonograms, and array and three-component analysis), (2) it is easy to use and reliable, (3) it has a macro programming language that allows users to develop innovative new analysis techniques, (4) it has interfaces to the Unix operating system, Matlab (www.mathworks.com), and the Generic Mapping Tools (GMT) software (Wessel and Smith, 1991, 1998 and gmt.soest.hawaii.edu) that make it very flexible, allowing researchers to solve many research problems innovatively with minimal programming effort, and (5) the suite of analysis capabilities are integrated so that innovative processing schemes are easily implemented. SAC is also widely used because of its user oriented development philosophy, which has led to consistent, easy to use capabilities that are backward compatible. A sample of some of SAC's capabilities is displayed in Figure 1.

  20. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. PMID:26567315

  1. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency.

  2. Bifunctional Homodimeric Triokinase/FMN Cyclase

    PubMed Central

    Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

    2014-01-01

    Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

  3. SAC-B, Argentine scientific satellite

    NASA Technical Reports Server (NTRS)

    Gulich, J. M.; White, C.

    1994-01-01

    The project and the missions of the Argentine scientific satellite, SAC-B, are summarized. SAC-B is an international cooperative project between NASA and the Secretariat of State of Science and Technology of the Argentine Republic. The objective of SAC-B is to advance the study of solar physics and astrophysics through the examination of solar flares, gamma ray burst sources and the diffuse soft X-ray cosmic background. The scientific payload comprises an instrument to measure the temporal evolution of X-ray emissions from solar flares as well as nonsolar gamma ray bursts, a combined soft X-ray and gamma ray burst detector, a diffuse X-ray background detector, and an energetic neutral atoms detector.

  4. A Surprise in the Lacrimal Sac

    PubMed Central

    Yuksel, Nilay; Akcay, Emine; Kilicarslan, Aydan; Ozen, Umut; Ozturk, Faruk

    2016-01-01

    To present a case with recurrent dacryocystitis as an unusual complication of medial orbital wall fracture repair with cartilage tissue graft. A 20-year-old male had facial trauma and underwent surgery to reconstruct right medial orbital wall fracture. During follow–up, he presented with continuous epiphora, mucopurulent discharge from the right eye. A thorough history taking indicated that medial orbital fracture was reconstructed with postauricular cartilage. We planned a standard external dacryocystorhinostomy (DCR). During the creation of lacrimal sac flaps, hard tissue was noted in the lacrimal sac. This tissue was excised and sent for pathological examination. The pathological examination revealed cartilage tissue. There were no further ipsilateral symptoms or complications after DCR. In patients with lacrimal system injury related to orbital wall fracture, iatrogenic foreign bodies in the lacrimal sac should be considered in patients with recurrent dacryocystitis who had reconstructive surgery for facial and orbital trauma. PMID:27555715

  5. Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

    PubMed Central

    Bender, Robert A.; Streicher, Stanley L.

    1979-01-01

    We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5′-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images PMID:33958

  6. Malignant melanoma of the lacrimal sac.

    PubMed

    Yamade, S; Kitagawa, A

    1978-01-01

    A case of malignant melanoma of the lacrimal sac in a 41-year-old woman is reported, which is propably the 12th one in the world literature. Dacryocystectomy is advisable at a localized stage. The importance of early diagnosis is discussed. PMID:714368

  7. Comparing and Contrasting NAEYC and SACS Accreditation

    ERIC Educational Resources Information Center

    Isaacs, Mary Ruth

    2010-01-01

    The purpose of this paper is to describe, compare, and contrast an early childhood departmental accreditation process from the National Association for the Education of Young Children (NAEYC) and a college/university-wide accreditation process from the Southern Association of Colleges and Schools (SACS). The history of higher education…

  8. 75. SAC control center underground structure middle floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    75. SAC control center underground structure middle floor plan, drawing number 32-02-03, dated 1 February, 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  9. 72. SAC control center underground structure lower floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    72. SAC control center underground structure lower floor plan, drawing number 32-02-03, dated 1 February 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  10. 77. SAC control center administrative section basement floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    77. SAC control center administrative section basement floor plan, drawing number not listed, dated 1 February, 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  11. 78. SAC control center aboveground addition partial first floor plan, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    78. SAC control center aboveground addition partial first floor plan, drawing number AW30-02-09, dated 15 October, 1962 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  12. 76. SAC control center underground structure upper floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    76. SAC control center underground structure upper floor plan, drawing number 32-02-03, dated 1 February, 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  13. 79. SAC control center administration section first floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    79. SAC control center administration section first floor plan, drawing number 32-02-03, dated 1 February, 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  14. 80. SAC control center administration section third floor plan, drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    80. SAC control center administration section third floor plan, drawing number 32-02-03, dated 1 February, 1955 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  15. 68. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    68. Aerial view of SAC command post, building 500, looking northeast, spring, 1957 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  16. 63. Aerial view of SAC command post construction, looking west ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    63. Aerial view of SAC command post construction, looking west - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  17. 67. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    67. Aerial view of SAC command post, building 500, looking northeast, undated - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  18. 62. Aerial view of SAC command post, building 500, looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    62. Aerial view of SAC command post, building 500, looking east - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  19. 3. Threequarter view of building 500 looking southeast from SAC ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Three-quarter view of building 500 looking southeast from SAC Boulevard - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  20. 13. SAC command center, weather center, underground structure, building 501, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. SAC command center, weather center, underground structure, building 501, undated - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Command Center, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  1. Surveillance Analysis Computer System (SACS) software requirements specification (SRS)

    SciTech Connect

    Glasscock, J.A.; Flanagan, M.J.

    1995-09-01

    This document is the primary document establishing requirements for the Surveillance Analysis Computer System (SACS) Database, an Impact Level 3Q system. The purpose is to provide the customer and the performing organization with the requirements for the SACS Project.

  2. Aneurysm sac shrinkage after endovascular treatment of the aorta: beyond sac pressure and endoleaks.

    PubMed

    Georgakarakos, Efstratios; Georgiadis, George S; Ioannou, Christos V; Kapoulas, Konstantinos C; Trellopoulos, George; Lazarides, Miltos

    2012-06-01

    The isolation of the aneurysm sac from systemic pressure and its consequent shrinkage are considered criteria of success after endovascular repair (EVAR). However, the process of shrinkage does not solely depend on the intrasac pressure, the predictive role of which remains ambiguous. This brief review summarizes the additional pathophysiological mechanisms that regulate the biomechanical properties of the aneurysm wall and may interfere with the process of aneurysm sac shrinkage. PMID:22402935

  3. Diterpene Cyclases and the Nature of the Isoprene Fold

    PubMed Central

    Cao, Rong; Zhang, Yonghui; Mann, Francis M.; Huang, Cancan; Mukkamala, Dushyant; Hudock, Michael P.; Mead, Matthew; Prisic, Sladjana; Wang, Ke; Lin, Fu-Yang; Chang, Ting-Kai; Peters, Reuben; Oldfield, Eric

    2013-01-01

    The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ∼20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there is an EDxxD-like Mg2+/diphosphate binding motif located in the γ-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. PMID:20602361

  4. What is inside the hernia sac?

    PubMed Central

    Virgínia, Ana Araújo; Santos, Cláudia; Contente, Helena; Branco, Cláudia

    2016-01-01

    Most ovarian inguinal hernias occur in children and are frequently associated with congenital genitalia defects. The authors present the case of a multiparous 89-year-old woman, without any genitalia defect, who was brought to the emergency department with an irreducible inguinal hernia. The patient was proposed for emergency surgery during which we encountered an ovary and a fallopian tube inside the hernial sac. An oophorosalpingectomy and a Lichtenstein procedure were carried out and the postoperative period was uneventful. This case shows that, even though it is rare, a hernial sac may contain almost any intra-abdominal organ, including those least frequent such as the appendix, an ovary or the fallopian tubes. PMID:27511751

  5. Aquarius/SAC-D Mission Overview

    NASA Technical Reports Server (NTRS)

    Sen, Amit; Kim, Yunjin; Caruso, Daniel; Lagerloef, Gary; Colomb, Raul; Yueh, Simon; LeVine, David

    2006-01-01

    Aquarius/SAC-D is a cooperative international mission developed between the National Aeronautics and Space Administration (NASA) of United States of America (USA) and the Comision Nacional de Actividades Espaciales (CONAE) of Argentina. The overall mission objective is to contribute to the understanding of the total Earth system and the consequences of the natural and man-made changes in the environment of the planet. Major themes are: ocean surface salinity, water cycle, climate, natural hazards and cryosphere.

  6. Membrane guanylyl cyclase receptors: an update

    PubMed Central

    Garbers, David L.; Chrisman, Ted D.; Wiegn, Phi; Katafuchi, Takeshi; Albanesi, Joseph P.; Bielinski, Vincent; Barylko, Barbara; Redfield, Margaret M.; Burnett, John C.

    2007-01-01

    Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance. PMID:16815030

  7. Periocular dirofilariasis mimicking lacrimal sac mucocoele.

    PubMed

    Pauly, Marian; Biswas, Jyotirmay; Hussain, Rameez N; Anantharaman, Giridhar

    2013-10-01

    Dirofilariasis is a zoonotic infection caused by filarial nematodes belonging to the genus dirofilariae. Dirofilaria is commonly seen in dogs, cats and other carnivorous animals world wide. Mosquitoes of the genus Culex, Anopheles and Aedes are the vectors and the humans are either incidental hosts or dead-end hosts. It affects lungs, liver and other visceral organs. Ocular involvement is rarely been reported. We present a case of 51-year-old female from Kerala, the southern State of India presented with a mass mimicking lacrimal sac mucocoele whose biopsy is proved to be dirofilariasis.

  8. Primary Nasopharngeal Yolk Sac Tumor: A Case Report.

    PubMed

    Arumugam, Dhanalakshmi; Thandavarayan, Pavithra; Chidambaram, Lalitha; Boj, Sudha; Marudasalam, Sumathi

    2016-05-01

    Yolk sac tumour also known as primitive endodermal tumour is the most common malignant germ cell tumour (GCT) in the paediatric age group. Most common sites of involvement are ovaries and testes, but rarely can occur in the extragonadal sites. In the head and neck region, yolk sac tumours have been reported in the nasopharynx, sinonasal tract, orbit, ear and parotid gland. Nasopharynx is an uncommon site for yolk sac tumour and very few cases of nasopharngeal pure yolk sac tumour have been reported so far. Yolk sac tumours are highly malignant and have a poor prognosis. This is a case of pure GCT in a three-year-old female child who presented with a rapidly growing nasopharyngeal mass. Histopathological examination followed by immunohistochemistry and serum AFP study clinched the diagnosis of yolk sac tumour. The tumour responded well to chemotherapy as evidenced by decrease in serum AFP levels. PMID:27437234

  9. Specific lignin accumulation in granulated juice sacs of Citrus maxima.

    PubMed

    Wu, Jia-Ling; Pan, Teng-Fei; Guo, Zhi-Xiong; Pan, Dong-Ming

    2014-12-17

    Juice sac granulation occurring in pummelo fruits [Citrus maxima (Burm.) Merr.] is an undesirable trait, and the underlying mechanism remains unresolved. Previous studies have shown that lignin metabolism is closely associated with the process of juice sac granulation. Here, a method suitable for lignin isolation from pummelo tissues is established. Acetylated lignins from different pummelo tissues and cultivars were analyzed by HSQC NMR. The results showed that lignins in granulated juice sacs were characterized by an extremely high abundance of guaiacyl units (91.13-96.82%), in contrast to lignins from other tissues, including leaves, stems, and segment membranes. The abnormally accumulated lignins in granulated juice sacs were specific and mainly polymerized from coniferyl alcohol. No significant difference was found in lignin types among various cultivars. These findings indicated that the mechanism of juice sac granulation might be similar among various cultivars, although very different degrees of juice sac granulation can be observed.

  10. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

    Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase

  11. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

    Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase

  12. Whisker Formation on SAC305 Soldered Assemblies

    NASA Astrophysics Data System (ADS)

    Meschter, S.; Snugovsky, P.; Bagheri, Z.; Kosiba, E.; Romansky, M.; Kennedy, J.; Snugovsky, L.; Perovic, D.

    2014-11-01

    This article describes the results of a whisker formation study on SAC305 assemblies, evaluating the effects of lead-frame materials and cleanliness in different environments: low-stress simulated power cycling (50-85°C thermal cycling), thermal shock (-55°C to 85°C), and high temperature/high humidity (85°C/85% RH). Cleaned and contaminated small outline transistors, large leaded quad flat packs (QFP), plastic leaded chip carrier packages, and solder balls with and without rare earth elements (REE) were soldered to custom designed test boards with Sn3Ag0.5Cu (SAC305) solder. After assembly, all the boards were cleaned, and half of them were recontaminated (1.56 µg/cm2 Cl-). Whisker length, diameter, and density were measured. Detailed metallurgical analysis on components before assembly and on solder joints before and after testing was performed. It was found that whiskers grow from solder joint fillets, where the thickness is less than 25 µm, unless REE was present. The influence of lead-frame and solder ball material, microstructure, cleanliness, and environment on whisker characteristics is discussed. This article provides detailed metallurgical observations and select whisker length data obtained during this multiyear testing program.

  13. Progressive myoclonus epilepsy associated with SACS gene mutations.

    PubMed

    Nascimento, Fábio A; Canafoglia, Laura; Aljaafari, Danah; Muona, Mikko; Lehesjoki, Anna-Elina; Berkovic, Samuel F; Franceschetti, Silvana; Andrade, Danielle M

    2016-08-01

    Pathogenic variants in the SACS gene (OMIM #604490) cause autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). ARSACS is a neurodegenerative early-onset progressive disorder, originally described in French Canadians, but later observed elsewhere.(1) Whole-exome sequencing of a large group of patients with unclassified progressive myoclonus epilepsies (PMEs) identified 2 patients bearing SACS gene mutations.(2) We detail the PME clinical features associated with SACS mutations and suggest the inclusion of the SACS gene in diagnostic screening of PMEs. PMID:27433545

  14. The triterpene cyclase protein family: a systematic analysis.

    PubMed

    Racolta, Silvia; Juhl, P Benjamin; Sirim, Demet; Pleiss, Jürgen

    2012-08-01

    Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction.

  15. Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors.

    PubMed Central

    Hayashi, F; Yamazaki, A

    1991-01-01

    Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors. Images PMID:1675787

  16. Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity.

    PubMed

    Arnaud, M; Vary, P; Zagorec, M; Klier, A; Debarbouille, M; Postma, P; Rapoport, G

    1992-05-01

    The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity. PMID:1577686

  17. On the robustness of SAC silencing in closed mitosis

    NASA Astrophysics Data System (ADS)

    Ruth, Donovan; Liu, Jian

    Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

  18. Fibrous dysplasia-like tumor of the lacrimal sac.

    PubMed

    Scott, Garrett R; Frueh, Bartley R; Flint, Andrew; Elner, Victor M

    2008-01-01

    A 75-year-old woman developed epiphora and a slowly enlarging right medial canthal mass for 1 year. CT revealed a mass with ground-glass radiodensity and hazy borders in the lacrimal sac. At external dacryocystorhinostomy, a tan, gritty, spherical mass was easily removed from the sac lumen. Histopathologic characteristics were typical of fibrous dysplasia. The postoperative course was uneventful.

  19. Laparoscopic management of hydatid cyst in the lesser sac

    PubMed Central

    Sahoo, Manash Ranjan; Kumar, Saurabh; Panda, Srikanta; Shameel, P. Ahammed

    2016-01-01

    Hydatid cyst is a disease caused by Echinococcus granulosus. Various anatomical location of hydatid cyst has been described in literature. Liver is the most common site of hydatid cyst and lungs are the second most common site. Hydatid cyst of lesser sac is a rare entity. Here we present a rare case report of laparoscopic management of hydatid cyst in lesser sac. PMID:27073313

  20. Differential air sac pressures in diving tufted ducks Aythya fuligula.

    PubMed

    Boggs, D F; Butler, P J; Wallace, S E

    1998-09-01

    The air in the respiratory system of diving birds contains a large proportion of the body oxygen stores, but it must be in the lungs for gas exchange with blood to occur. To test the hypothesis that locomotion induces mixing of air sac air with lung air during dives, we measured differential pressures between the interclavicular and posterior thoracic air sacs in five diving tufted ducks Aythya fuligula. The peak differential pressure between posterior thoracic and interclavicular air sacs, 0.49+/-0.13 kPa (mean +/- s.d.), varied substantially during underwater paddling as indicated by gastrocnemius muscle activity. These data support the hypothesis that locomotion, perhaps through associated abdominal muscle activity, intermittently compresses the posterior air sacs more than the anterior ones. The result is differential pressure fluctuations that might induce the movement of air between air sacs and through the lungs during dives. PMID:9716518

  1. Soluble variants of human recombinant glutaminyl cyclase.

    PubMed

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of (1)H-(15)N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease. PMID:23977104

  2. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  3. Soluble Variants of Human Recombinant Glutaminyl Cyclase

    PubMed Central

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of 1H-15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease. PMID:23977104

  4. Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

    PubMed

    Takemoto, Kenji; Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-01

    Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

  5. Diuretics and the renal adenylate cyclase system

    PubMed Central

    Dawborn, J.K.; Macneil, S.; Martin, T.J.

    1977-01-01

    1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism. PMID:202362

  6. Bilateral congenital lacrimal sac mucoceles with nasal extension and drainage.

    PubMed

    Divine, R D; Anderson, R L; Bumsted, R M

    1983-02-01

    A newborn infant with bilateral mucoceles of the lacrimal sacs also had submucosal masses along the floor of the nose beneath the inferior turbinates communicating with the mucoceles. Drainage of the mucoceles was performed by needle aspiration and wide marsupialization of the nasal masses into the nose under direct visualization. To our knowledge, this is the first time that intranasal extension of mucoceles has been reported, and the first time that lacrimal sac mucoceles have been successfully treated via direct nasal drainage. We advocate careful nasal evaluation in cases of congenital lacrimal sac mucoceles to determine whether intranasal extension is common and whether intranasal drainage can be curative.

  7. Farnesyloxycoumarins, a new class of squalene-hopene cyclase inhibitors.

    PubMed

    Cravotto, Giancarlo; Balliano, Gianni; Robaldo, Bruna; Oliaro-Bosso, Simonetta; Chimichi, Stefano; Boccalini, Marco

    2004-04-19

    A few naturally occurring prenyl- and prenyloxycoumarins and several new related synthetic derivatives were evaluated as inhibitors of squalene-hopene cyclase (SHC), a useful model enzyme, to predict their interactions with oxidosqualene cyclase (OSC). Umbelliprenin-10',11'-monoepoxide (IC(50) 2.5 microM) and the corresponding 6',7'-10',11' diepoxide (IC(50) 1.5 microM) were the most active enzyme inhibitors.

  8. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  9. 79. Sac digital network (Sacdin), summary fault indicator at top, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    79. Sac digital network (Sacdin), summary fault indicator at top, south side - Ellsworth Air Force Base, Delta Flight, Launch Control Facility, County Road CS23A, North of Exit 127, Interior, Jackson County, SD

  10. A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.

    PubMed

    Sanyal, Anwesha; Chen, Andy J; Nakayasu, Ernesto S; Lazar, Cheri S; Zbornik, Erica A; Worby, Carolyn A; Koller, Antonius; Mattoo, Seema

    2015-03-27

    The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling. PMID:25601083

  11. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.

  12. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record. PMID:17686016

  13. Automatic segmentation and classification of gestational sac based on mean sac diameter using medical ultrasound image

    NASA Astrophysics Data System (ADS)

    Khazendar, Shan; Farren, Jessica; Al-Assam, Hisham; Sayasneh, Ahmed; Du, Hongbo; Bourne, Tom; Jassim, Sabah A.

    2014-05-01

    Ultrasound is an effective multipurpose imaging modality that has been widely used for monitoring and diagnosing early pregnancy events. Technology developments coupled with wide public acceptance has made ultrasound an ideal tool for better understanding and diagnosing of early pregnancy. The first measurable signs of an early pregnancy are the geometric characteristics of the Gestational Sac (GS). Currently, the size of the GS is manually estimated from ultrasound images. The manual measurement involves multiple subjective decisions, in which dimensions are taken in three planes to establish what is known as Mean Sac Diameter (MSD). The manual measurement results in inter- and intra-observer variations, which may lead to difficulties in diagnosis. This paper proposes a fully automated diagnosis solution to accurately identify miscarriage cases in the first trimester of pregnancy based on automatic quantification of the MSD. Our study shows a strong positive correlation between the manual and the automatic MSD estimations. Our experimental results based on a dataset of 68 ultrasound images illustrate the effectiveness of the proposed scheme in identifying early miscarriage cases with classification accuracies comparable with those of domain experts using K nearest neighbor classifier on automatically estimated MSDs.

  14. The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

    PubMed

    Jiang, P; Peliska, J A; Ninfa, A J

    1998-09-15

    The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed. PMID:9737857

  15. Protein kinase C sensitizes olfactory adenylate cyclase

    PubMed Central

    1993-01-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  16. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. PMID:27030537

  17. Intraoperative Sac Pressure Measurement During Endovascular Abdominal Aortic Aneurysm Repair

    SciTech Connect

    Ishibashi, Hiroyuki; Ishiguchi, Tsuneo; Ohta, Takashi; Sugimoto, Ikuo; Iwata, Hirohide; Yamada, Tetsuya; Tadakoshi, Masao; Hida, Noriyuki; Orimoto, Yuki; Kamei, Seiji

    2010-10-15

    PurposeIntraoperative sac pressure was measured during endovascular abdominal aortic aneurysm repair (EVAR) to evaluate the clinical significance of sac pressure measurement.MethodsA microcatheter was placed in an aneurysm sac from the contralateral femoral artery, and sac pressure was measured during EVAR procedures in 47 patients. Aortic blood pressure was measured as a control by a catheter from the left brachial artery.ResultsThe systolic sac pressure index (SPI) was 0.87 {+-} 0.10 after main-body deployment, 0.63 {+-} 0.12 after leg deployment (P < 0.01), and 0.56 {+-} 0.12 after completion of the procedure (P < 0.01). Pulse pressure was 55 {+-} 21 mmHg, 23 {+-} 15 mmHg (P < 0.01), and 16 {+-} 12 mmHg (P < 0.01), respectively. SPI showed no significant differences between the Zenith and Excluder stent grafts (0.56 {+-} 0.13 vs. 0.54 {+-} 0.10, NS). Type I endoleak was found in seven patients (15%), and the SPI decreased from 0.62 {+-} 0.10 to 0.55 {+-} 0.10 (P = 0.10) after fixing procedures. Type II endoleak was found in 12 patients (26%) by completion angiography. The SPI showed no difference between type II endoleak positive and negative (0.58 {+-} 0.12 vs. 0.55 {+-} 0.12, NS). There were no significant differences between the final SPI of abdominal aortic aneurysms in which the diameter decreased in the follow-up and that of abdominal aortic aneurysms in which the diameter did not change (0.53 {+-} 0.12 vs. 0.57 {+-} 0.12, NS).ConclusionsSac pressure measurement was useful for instant hemodynamic evaluation of the EVAR procedure, especially in type I endoleaks. However, on the basis of this small study, the SPI cannot be used to reliably predict sac growth or regression.

  18. A novel adenylylation process in liver plasma membrane-bound proteins

    SciTech Connect

    San Jose, E.; Benguria, A.; Villalobo, A. )

    1990-11-25

    Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with (alpha-32P)ATP as well as with (gamma-32P)ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue (alpha, beta-methylene)ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with (alpha-32P)ATP are glycoproteins bound to the cell plasma membrane.

  19. Nicotinamide Mononucleotide Adenylyl Transferase 2: A Promising Diagnostic and Therapeutic Target for Colorectal Cancer

    PubMed Central

    Cui, Chunhui; Qi, Jia; Deng, Quanwen; Chen, Rihong; Zhai, Duanyang; Yu, Jinlong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers all over the world. It is essential to search for more effective diagnostic and therapeutic methods for CRC. Abnormal nicotinamide adenine dinucleotide (NAD) metabolism has been considered as a characteristic of cancer cells. In this study, nicotinamide mononucleotide adenylyl transferases (NMNATs) as well as p53-mediated cancer signaling pathways were investigated in patients with colorectal cancer. The CRC tissues and adjacent normal tissues were obtained from 95 untreated colorectal cancer patients and were stained for expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and p53. The survival rate was analyzed by the Kaplan-Meier method and the log-rank test. The multivariate Cox proportional hazard regression analysis was conducted as well. Our data demonstrated that expression of NMNAT2 and p53 was significantly higher in CRC tissues, while NMNAT2 expression is in correlation with the invasive depth of tumors and TNM stage. Significant positive correlation was found between the expression of NMNAT2 and the expression of p53. However, NMNAT2 expression was not a statistically significant prognostic factor for overall survival. In conclusion, our results indicated that NMNAT2 might participate in tumorigenesis of CRC in a p53-dependent manner and NMNAT2 expression might be a potential therapeutic target for CRC. PMID:27218101

  20. The anuran vocal sac: a tool for multimodal signalling.

    PubMed

    Starnberger, Iris; Preininger, Doris; Hödl, Walter

    2014-11-01

    Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems.

  1. The anuran vocal sac: a tool for multimodal signalling

    PubMed Central

    Starnberger, Iris; Preininger, Doris; Hödl, Walter

    2014-01-01

    Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems. PMID:25389375

  2. The anuran vocal sac: a tool for multimodal signalling.

    PubMed

    Starnberger, Iris; Preininger, Doris; Hödl, Walter

    2014-11-01

    Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems. PMID:25389375

  3. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst. PMID:22526778

  4. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

  5. Aquarius and the Aquarius/SAC-D Mission

    NASA Technical Reports Server (NTRS)

    LeVine, D. M.; Lagerloef, G. S. E.; Torrusio, S.

    2010-01-01

    Aquarius is a combination L-band radiometer and scatterometer designed to map the salinity field at the ocean surface from space. It will be flown on the Aquarius/SAC-D mission, a partnership between the USA space agency (NASA) and Argentine space agency (CONAE). The mission is composed of two parts: (a) The Aquarius instrument being developed as part of NASA.s Earth System Science Pathfinder (ESSP) program; and (b) SAC-D the fourth spacecraft service platform in the CONAE Satellite de Aplicaciones Cientificas (SAC) program. The primary focus of the mission is to monitor the seasonal and interannual variations of the salinity field in the open ocean. The mission also meets the needs of the Argentine space program for monitoring the environment and for hazard detection and includes several instruments related to these goals.

  6. [Spanish adaptation of Hobfoll's Strategic Approach to Coping Scale (SACS)].

    PubMed

    Pedrero Pérez, Eduardo J; Santed Germán, Miguel A; Pérez García, Ana M

    2012-01-01

    The present research adapted the Strategic Approach to Coping Scale (SACS), developed by Hobfoll and colleagues, to the Spanish population. SACS is an instrument derived from Hobfoll's Conservation of Resources Theory, which emphasises the contribution of social factors to coping processes. This instrument assesses coping strategies in 9-subscales, organised in three dimensions: orientation to the problem (active/passive), use of social resources (prosocial/antisocial), and orientation to others involved (direct/indirect). The Spanish version, administered to a non-clinical sample (N= 767), found 7-subscales structured in prosocial/antisocial, active/passive and reflexive/intuitive dimensions, with adequate reliability and construct validity. To conclude, the Spanish SACS is a potentially useful and reliable instrument for research and clinical purposes, mainly in areas in which social components need to be explicitly considered.

  7. Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells.

    PubMed

    Bresson-Bépoldin, L; Jacquot, M C; Schlegel, W; Rawlings, S R

    1998-10-01

    Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology. PMID:9801454

  8. Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells.

    PubMed

    Bresson-Bépoldin, L; Jacquot, M C; Schlegel, W; Rawlings, S R

    1998-10-01

    Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology.

  9. Region-Specific Disruption of Adenylate Cyclase Type 1 Gene Differentially Affects Somatosensorimotor Behaviors in Mice(1,2,3).

    PubMed

    Arakawa, Hiroyuki; Akkentli, Fatih; Erzurumlu, Reha S

    2014-01-01

    Cover FigureRegion-specific adenylyl cyclase 1 (AC1) loss of function differentially affects both patterning and sensorimotor behaviors in mice. AC1 is expressed at all levels of the somatosensory pathway and plays a major role in refinement and patterning of topographic sensory maps. Cortex-specific AC1 loss of function (CxAC1KO mice) does not affect barrel patterning and activation of specific barrels corresponding to stimulated whiskers and does not impair sensorimotor behaviors. While global (AC1KO) and thalamus-specific (ThAC1KO) AC1 loss of function leads to absence of barrel patterns, selective whisker stimulation activates topographically aligned cortical loci. Despite functional topography of the whisker-barrel cortex, sensorimotor and social behaviors are impaired, indicating the importance of patterning of topographical sensory maps in the neocortex. Adenylate cyclase type I (AC1) is primarily, and, abundantly, expressed in the brain. Intracellular calcium/calmodulin increases regulate AC1 in an activity-dependent manner. Upon stimulation, AC1 produces cAMP and it is involved in the patterning and the refinement of neural circuits. In mice, spontaneous mutations or targeted deletion of the Adcy1 gene, which encodes AC1, resulted in neuronal pattern formation defects. Neural modules in the primary somatosensory (SI) cortex, the barrels, which represent the topographic distribution of the whiskers on the snout, failed to form (Welker et al., 1996; Abdel-Majid et al., 1998). Cortex- or thalamus-specific Adcy1 deletions led to different cortical pattern phenotypes, with thalamus-specific disruption phenotype being more severe (Iwasato et al., 2008; Suzuki et al., 2013). Despite the absence of barrels in the "barrelless"/Adcy1 null mice, thalamocortical terminal bouton density and activation of cortical zones following whisker stimulation were roughly topographic (Abdel-Majid et al., 1998; Gheorghita et al., 2006). To what extent does patterning of the

  10. Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

    PubMed Central

    Reynaldo, L. P.; Villafranca, J. J.; Horrocks, W. D.

    1996-01-01

    Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation

  11. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

    PubMed

    Susick, Laura L; Lowing, Jennifer L; Bosse, Kelly E; Hildebrandt, Clara C; Chrumka, Alexandria C; Conti, Alana C

    2014-08-01

    Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the

  12. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure

    PubMed Central

    Susick, Laura L.; Lowing, Jennifer L.; Bosse, Kelly E.; Hildebrandt, Clara C.; Chrumka, Alexandria C.; Conti, Alana C.

    2014-01-01

    Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the

  13. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  14. Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

    PubMed

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

  15. General base-general acid catalysis by terpenoid cyclases.

    PubMed

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  16. Soil Moisture Retrieval Using the Aquarius/SAC-D Instruments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aquarius/SAC-D will share common elements with several current and future satellite missions that provide soil moisture. Passive microwave soil moisture retrieval using low frequencies is currently performed using Aqua Advanced Microwave Scanning Radiometer-E (AMSR-E) (C/X-band). This will extended ...

  17. Placentation in mammals: Definitive placenta, yolk sac, and paraplacenta.

    PubMed

    Carter, A M; Enders, A C

    2016-07-01

    An overview is given of variations in placentation with particular focus on yolk sac, paraplacenta, and other structures important to histotrophic nutrition. The placenta proper varies in general shape, internal structure, and the number of tissues in the interhemal barrier. Yolk sac membranes persist to term in insectivores, colugos, rodents, and lagomorphs. In the latter two orders, they are of known importance for maternal-fetal transfer of antibodies, vitamins, lipids, and proteins. The detached yolk sac of bats is also active throughout gestation. A vascular paraplacenta, or smooth chorioallantois, has known functions in ruminants and carnivores and is found in several other orders of mammal where its function has yet to be explored. In human gestation, the chorion (avascular chorioallantois) is important for hormone synthesis. The true chorion of squirrels and hedgehogs is avascular but may nevertheless allow transfer from mother to fetus through the exocelom. Hemophagous areas with columnar trophoblast are paraplacental structures in carnivores and elephants but occur also within the placenta as in hyenas and moles. In shrews, it is the yolk sac that ingests and processes red cells. Areolas and chorionic vesicles are other structures important for absorption of uterine secretions and ingestion of cellular debris. In conclusion, we find that paraplacental structures, while showing less variation than the placenta proper, contribute not just to the integrity of overall placentation, but in various ways to maternal-fetal interrelationships. PMID:27155730

  18. The annular hematoma of the shrew yolk-sac placenta.

    PubMed

    King, B F; Enders, A C; Wimsatt, W A

    1978-05-01

    The annular hematoma of the shrew, Blarina brevicauda, is a specialized portion of the yolk-sac wall. In this study, we have examined the fine structure of the different cellular components of the anular hematoma. Small pieces of the gestation sacs from seven pregnant shrews were fixed in glutaraldehyde and osmium tetroxide and processed for transmission electron microscopy. In the area of the trophoblastic curtain, the maternal capillary endothelial cells were hypertrophied and syncytial trophoblast surrounded the capillaries. Cellular trophoblast covered part of the luminal surface of the curtain region, whereas masses of apparently degenerating syncytium were present on other areas of the surface. Maternal erythrocytes, released into the uterine lumen from the curtain region, were phagocytized and degraded by the columnar cells of the trophoblastic annulus. No evidence of iron or pigment accumulation was evident in the parietal endodermal cells underlying the annular trophoblast. Parietal endodermal cells were characterized by cuboidal shape, widely dilated intercellular spaces, and cytoplasm containing granular endoplasmic reticulum. Endodermal cells of the visceral yolk-sac accumulated large numbers of electron-dense granules as well as glycogen in their cytoplasm. Hemopoietic areas and vitelline capillaries were found subjacent to the visceral endoderm. The various portions of the yolk-sac wall of Blarina appear to perform complementary functions which are probably important in maternal-fetal iron transfer. PMID:677046

  19. TMACS Test Procedure TP008: SACS Interface. Revision 5

    SciTech Connect

    Washburn, S.J.

    1994-05-31

    The TMACS Software Project Test Procedures translate the project`s acceptance criteria into test steps. Software releases are certified when the affected Test Procedures are successfully performed and the customers authorize installation of these changes. This Test Procedure tests the TMACS SACS Interface functions.

  20. Efficacy of Several Pesticide Products on Brown Widow Spider (Araneae: Theridiidae) Egg Sacs and Their Penetration Through the Egg Sac Silk.

    PubMed

    Vetter, Richard S; Tarango, Jacob; Campbell, Kathleen A; Tham, Christine; Hayashi, Cheryl Y; Choe, Dong-Hwan

    2016-02-01

    Information on pesticide effects on spiders is less common than for insects; similar information for spider egg sacs is scarcer in the open literature. Spider egg sacs are typically covered with a protective silk layer. When pesticides are directly applied to egg sacs, the silk might prevent active ingredients from reaching the eggs, blocking their insecticidal effect. We investigated the impact of six water-based pesticide sprays and four oil-based aerosol products against egg sacs of brown widow spiders, Latrodectus geometricus C. L. Koch. All water-based spray products except one failed to provide significant mortality to egg sacs, resulting in successful spiderling emergence from treated egg sacs at a similar rate to untreated egg sacs. In contrast to water-based sprays, oil-based aerosols provided almost complete control, with 94-100% prevention of spiderling emergence. Penetration studies using colored pesticide products indicated that oil-based aerosols were significantly more effective in penetrating egg sac silk than were the water-based sprays, delivering the active ingredients on most (>99%) of the eggs inside the sac. The ability of pesticides to penetrate spider egg sac silk and deliver lethal doses of active ingredients to the eggs is discussed in relation to the chemical nature of egg sac silk proteins. Our study suggests that pest management procedures primarily relying on perimeter application of water-based sprays might not provide satisfactory control of brown widow spider eggs. Determination of the most effective active ingredients and carrier characteristics warrant further research to provide more effective control options for spider egg sacs.

  1. Investigation of sulfonated aromatic compound (SAC) modification to nylon film. 2. Study of SAC sorption isotherm and atomic force microscopic characterization of nylon surfaces

    SciTech Connect

    Zhang, J.; Watson, B.A.; Keown, R.W.; Malone, C.P.; Barteau, M.A.

    1995-08-01

    Nylon 6 and nylon 66 films have been treated with aqueous sulfonated aromatic compound (SAC) solutions at concentrations ranging from 0.005 to 1.0 wt%. SAC uptakes at different treatment concentrations were measured and found to follow a BET isotherm. The surface morphologies of nylon film samples, including the original and SAC-treated films, have been characterized by atomic force microscopy (AFM). For untreated nylon 6 and nylon 66 films, AFM images show a randomly distributed fibrillar surface structure. Characteristic widths of fibrils in the nylon 66 and 6 films were 150-225 and 75-150 nm, respectively. For SAC-treated nylon films, the AFM images revealed that the surfaces of the films became covered with nodule-like features having a diameter range of 25-60 nm. AFM analysis provides evidence that SAC treatment deposited a surface coating on nylon films. AFM images of SAC-treated nylon films suggest a mechanism for stain resistance in which the SAC first forms a thin coating on the nylon via bondings between attractive groups in the SAC and nylon polymers. After treatment at increased SAC concentration, the surface is covered with nodule-like deposits which likely serve as a physical barrier to dye permeation. 20 refs., 8 figs.

  2. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  3. Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Rhodopseudomonas sphaeroides.

    PubMed

    Wang, X; Song, H Y

    1989-08-01

    Following a freeze-thaw cycle, and the treatment of Rhodopseudomonas sphaeroides with the nonionic detergent Lubrol PX, the permeabilized cell suspensions can be assayed directly both for the intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number n of adenylylated subunits/dodecameric molecules). It seems that all components of the bicycle system are retained if cells grown with low concentrations of ammonia as the sole nitrogen source are used. The value of n was dependent upon the concentration of substrates (ATP, Pi) and allosteric effectors (ATP, glutamine and alpha-ketoglutarate) of adenylytransferase. The value of n affected by UTP, the specific substrate of the uridylyltransferase shows first the evidence that the bicycle cascade control system studied in Escherichia coli may exist in this phototrophic bacterium. PMID:2575389

  4. Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis

    PubMed Central

    Luitz, Manuel P.; Bomblies, Rainer; Ramcke, Evelyn; Itzen, Aymelt; Zacharias, Martin

    2016-01-01

    The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the active Rab1b state by locking the switch in the active signaling conformation independent of bound GTP or GDP and that electrostatic interactions due to the additional negative charge in the switch region make significant contributions. The stacking interaction between adenine and Phe45 however, seems to have only minor influence on this stabilisation. The results may also have implications for the mechanistic understanding of conformational switching in other signaling proteins. PMID:26818796

  5. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explain observed abnormalities of phosphorylase activation and sarcoplasmic reticulum function. PMID:6271806

  6. Engineering adenylate cyclases regulated by near-infrared window light

    PubMed Central

    Ryu, Min-Hyung; Kang, In-Hye; Nelson, Mathew D.; Jensen, Tricia M.; Lyuksyutova, Anna I.; Siltberg-Liberles, Jessica; Raizen, David M.; Gomelsky, Mark

    2014-01-01

    Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. PMID:24982160

  7. [Adenocarcinoma of the endolymphatic sac in children. A case report].

    PubMed

    Facon, F; Bruzzo, M; Figarella-Branger, D; Girard, N; Chays, A; Magnan, J

    2002-01-01

    We report a case of an adenocarcinoma of the endolymphatic sac in a 13 years old child, which was fortuitously discovered at the time of radiological investigation of growth delay. Arising from the end of the endolymphatic sac, this rare tumour shares a similar clinic expression with other cerebellopontine angle pathologies. Radiological investigation reveals a moth eaten appearance to the posterior temporal bone and specific bony inclusions. With MRI there is an intense and/or intermediate signal on T1, a hyper signal on T2 and T1 enhancement with injection of gadolinium. Treatment is surgical and local recurrences are not uncommon. After histological confirmation, a search for Von Hippel-Lindau disease is mandatory because this tumour is not exceptional among this subset of patients. Diagnosis and therapeutic criteria will be highlighted by our case and a review of the literature.

  8. Development of yolk sac inversion in Galea spixii and Cavia porcellus (Rodentia, Caviidae).

    PubMed

    de Oliveira, M F; do Vale, A M; Favaron, P O; Vasconcelos, B G; de Oliveira, G B; Miglino, M A; Mess, A

    2012-10-01

    Caviomorph development includes an inverted yolk sac. Since principle processes are not understood, we investigated its differentiation in Galea and re-examined material from the guinea pig. Galea showed the typical caviomorph conditions in blastocyst development and the nature of the definitive yolk sac, formed of the visceral layer that became villous, proliferative, vascularized and attached to the uterus and placenta. In contrast to what was known before, in both species parts of the parietal yolk sac and a yolk sac cavity were temporarily present. Data suggest that early yolk sac development in caviomorphs is more complex than thought before.

  9. Reformatted planar 'Christmas tree' MR appearance of the endolymphatic sac.

    PubMed

    Oehler, M C; Chakeres, D W; Schmalbrock, P

    1995-08-01

    A high-resolution three-dimensional Fourier transform technique and prototype bilateral dual phased-array surface coil technique was used to make inner ear structures visible on MR. Multiplanar reformatted images, parallel to the plane of the vestibular aqueduct, allowed viewing of the entire endolymphatic sac/vestibular aqueduct on one section, producing a "Christmas tree" shape. The reformation was obtained using a double oblique angle, 45 degrees from true sagittal and 70 degrees from the orbital-meatal axis.

  10. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  11. Endoscopic examination of snakes by access through an air sac.

    PubMed

    Jekl, V; Knotek, Z

    2006-03-25

    Sixteen boa constrictors (Boa constrictor), three royal pythons (Python regius) and 15 Burmese pythons (Python molurus bivittatus) were examined endoscopically by access through the air sac. The snakes were immobilised in a ventral position using a half-open anaesthetic system with assisted ventilation and a mixture of isoflurane and oxygen. The rigid endoscope was introduced percutaneously and the internal structure of the lungs and the air sac, and the shape, size and external surface of the liver were visible in the cranial direction. In the smaller snakes the bifurcation and caudal part of the trachea could be viewed, provided the endoscope was positioned in a retrograde orientation. The caudal orientation of the endoscope made it possible to view the gall bladder and the size, shape and surface of the spleen. In some cases, the pancreas and the surface of the stomach and colon could be monitored. Endoscopy through the air sac also made it possible to check the major veins in the coelom. The snakes were monitored for at least 30 days after the intervention and no changes in their respiratory function or general health were observed.

  12. Solitary fibrous tumors arising in abdominal wall hernia sacs.

    PubMed

    Lee, J R; Hancock, S M; Martindale, R G

    2001-06-01

    Solitary fibrous tumor (SFT) of the peritoneum is an unusual spindle-cell neoplasm. SFT was originally described in the pleura; however it is now diagnosed in multiple extrathoracic sites. Most believe that the tumor is of mesenchymal origin and should be classified as a variant of fibroma. SFT of the pleura and peritoneum have also been called fibrous mesothelioma, and the cell of origin is felt to be a pluripotential submesothelial mesenchymal cell. Primary tumors arising in hernia sacs are rare, and we report on two patients with hernia SFT. The first is a 67-year-old man who had a diffusely thickened distal left inguinal hernia sac. Within the sac was copious myxoid material mimicking pseudomyxoma peritonei. Herniorrhaphy and orchiectomy were performed. The second is a 44-year-old woman with a midepigastric mass attached to a ventral hernia. Wide local excision was performed. Both tumors demonstrated plump spindle cells, one with myxoid background and the other with keloidal collagen. Calretinin immunostaining was positive in both tumors, whereas CD34 was negative. This suggests tumor origin from a submesothial pluripotential cell that maintains potential for mesothelial differentiation. Surgical excision is the treatment of choice with the degree of resectability being a powerful predictor of outcome.

  13. Carcinoma of the anal sac glands in ranch mink.

    PubMed

    Hadlow, W J

    1985-05-01

    During a 14-year period, carcinoma of the anal sac apocrine glands was found in 52 pastel and 8 sapphire mink (Mustela vison) kept for studies on slow viral diseases. The pastel mink varied in age from 72 to 135 months (mean age 108 months), the sapphire mink from 63 to 100 months (mean age 81 months). All but one pastel mink were females. The primary tumor varied in size from masses that caused bulges in the perineum to those that were found only after microscopic examination of the anal sac glands. Although the primary tumor grew mainly by expansion with little local infiltration, 41 of the 60 tumors had metastasized to the regional lymph nodes and sometimes also to more distant sites. The striking propensity of the carcinoma to metastasize while still small, even microscopic, often resulted in massive secondary growths, notably in the iliac lymph nodes. Hypercalcemia did not accompany the carcinoma. Its varied microscopic appearance included solid, glandular, squamous cell, and spindle or round cell components. Combinations of them formed mixed or complex histologic patterns, no doubt largely attributable to neoplastic proliferation of myoepithelial cells and squamous metaplasia of the apocrine gland epithelium. Although its cause remains obscure, the carcinoma appeared to arise from small foci of hyperplastic apocrine glands, sometimes in relation to both anal sacs. The tumor is a common and distinctive expression of neoplasia in older ranch mink.

  14. Educational report: A case of lacrimal sac rhinosporidiosis.

    PubMed

    Jamison, Aaron; Crofts, Kevin; Roberts, Fiona; Gregory, Maria Elena

    2016-10-01

    This article reports a presentation of lacrimal sac rhinosporidiosis and informs the reader of this uncommon but important diagnosis. A 36-year-old man from Pakistan presented with a 3-month history of swelling at the nasal aspect of the left lower lid. This was associated with occasional crepitus and slight localised discomfort, but no epiphora. There was a palpable fullness near the left medial canthus associated with telecanthus but a normal sac washout and normal eye examination otherwise. Previous medical history included an ipsilateral nasal polypectomy and inferior meatal antrostomy around 10 years previously, whilst living in Pakistan. Various imaging modalities were useful in identifying a soft tissue mass within the left nasolacrimal duct. Following excision biopsy, histological examination confirmed the presence of rhinosporidiosis, likely caused by the organism Rhinosporidium seeberi. Rhinosporidiosis should be considered as a potential cause in any case of lacrimal sac pathology. Imaging studies may be helpful in measuring the extent of disease, although histological examination is required to confirm the diagnosis. Although rare, the complications of rhinosporidiosis can be potentially blinding or fatal. As discussed in this case, the presence of telecanthus may represent a lacrimal system tumour, either malignant or benign, and should always prompt further investigation. PMID:27541939

  15. Oblique sectional planes of block plastinates eased by Sac Plastination.

    PubMed

    Kürtül, Ibrahim; Hammer, Niels; Rabi, Suganthy; Saito, Toshiyuki; Böhme, Jörg; Steinke, Hanno

    2012-07-01

    To find an oblique cutting plane of a plastinate, e.g. to cut gamma-nails in the femur, the Block Plastination technique was modified. After CT and MRI examination, the specimens were plastinated with the standard resin mixture E6/E12/E600. Instead of using a box to form a block during the casting and curing stage, we embedded the specimen in a sac made of polyester foil. A polymerized wooden block was attached to the specimen. The sac was wrapped with tape to the embedded specimen with the block. This approach limited the amount of required resin to the inner volume of the plastinate. Then, the plastination sac was put in the incubator for further polymerization and curing. When the foil was removed from the plastinated specimen, the wooden block served as a socket for the grip when sawing. The outer shape of the specimen remained visible. Doing so, the adequate cutting plane could be determined easily. PMID:22209028

  16. Allosteric Activation of the Phosphoinositide Phosphatase Sac1 by Anionic Phospholipids

    PubMed Central

    2012-01-01

    Sac family phosphoinositide phosphatases comprise an evolutionarily conserved family of enzymes in eukaryotes. Our recently determined crystal structure of the Sac phosphatase domain of yeast Sac1, the founding member of the Sac family proteins, revealed a unique conformation of the catalytic P-loop and a large positively charged groove at the catalytic site. We now report a unique mechanism for the regulation of its phosphatase activity. Sac1 is an allosteric enzyme that can be activated by its product phosphatidylinositol or anionic phospholipid phosphatidylserine. The activation of Sac1 may involve conformational changes of the catalytic P-loop induced by direct binding with the regulatory anionic phospholipids in the large cationic catalytic groove. These findings highlight the fact that lipid composition of the substrate membrane plays an important role in the control of Sac1 function. PMID:22452743

  17. Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

    PubMed

    Wintjens, René; Belrhali, Hassan; Clantin, Bernard; Azarkan, Mohamed; Bompard, Coralie; Baeyens-Volant, Danielle; Looze, Yvan; Villeret, Vincent

    2006-03-24

    Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

  18. Sac Angiography and Glue Embolization in Emergency Endovascular Aneurysm Repair for Ruptured Abdominal Aortic Aneurysm

    SciTech Connect

    Koike, Yuya Nishimura, Jun-ichi Hase, Soichiro Yamasaki, Motoshige

    2015-04-15

    PurposeThe purpose of this study was to demonstrate a sac angiography technique and evaluate the feasibility of N-butyl cyanoacrylate (NBCA) embolization of the ruptured abdominal aortic aneurysm (AAA) sac in emergency endovascular aneurysm repair (EVAR) in hemodynamically unstable patients.MethodsA retrospective case series of three patients in whom sac angiography was performed during emergency EVAR for ruptured AAA was reviewed. After stent graft deployment, angiography within the sac of aneurysm (sac angiography) was performed by manually injecting 10 ml of contrast material through a catheter to identify the presence and site of active bleeding. In two patients, sac angiography revealed active extravasation of the contrast material, and NBCA embolization with a coaxial catheter system was performed to achieve prompt sealing.ResultsSac angiography was successful in all three patients. In the two patients who underwent NBCA embolization for aneurysm sac bleeding, follow-up computed tomography (CT) images demonstrated the accumulation of NBCA consistent with the bleeding site in preprocedural CT images.ConclusionsEVAR is associated with a potential risk of ongoing bleeding from type II or IV endoleaks into the disrupted aneurysm sac in patients with severe coagulopathy. Therefore, sac angiography and NBCA embolization during emergency EVAR may represent a possible technical improvement in the treatment of ruptured AAA in hemodynamically unstable patients.

  19. Rhinosporidiosis of lacrimal sac: An interesting case of orbital swelling

    PubMed Central

    Basu, Sandip Kanti; Bain, Jayanta; Maity, Kuntal; Chattopadhyay, Debarati; Baitalik, Debasis; Majumdar, Bijay Kumar; Gupta, Vivek; Kumar, Ashwini; Dalal, Bibhas Saha; Malik, Anu

    2016-01-01

    Rhinosporidiosis is a chronic localized granulomatous disease caused by Rhinosporidium seeberi, an aquatic protistan parasite belonging to a clade, Mesomycetozoea. Infestation of Rhinosporidiosis to the eye and adnexa is termed oculosporidiosis, in such cases, conjunctival mucosa is mostly involved; however in our case, it involved only the lacrimal sac and deeper periorbital tissue and presented as a case of orbital swelling. Surgical excision of the lesion was done, postoperatively dapsone therapy was given for 6 months, and the patient responded very well with no recurrence till date. PMID:27003980

  20. A new small molecule inhibitor of soluble guanylate cyclase

    PubMed Central

    Mota, Filipa; Gane, Paul; Hampden-Smith, Kathryn; Allerston, Charles K.; Garthwaite, John; Selwood, David L.

    2015-01-01

    Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay. PMID:26264842

  1. Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco.

    PubMed

    Hohn, T M; Ohlrogge, J B

    1991-09-01

    The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [(3)H]farnesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product. PMID:16668409

  2. Venous Sac Embolization of Pulmonary Arteriovenous Malformation: Preliminary Experience Using Interlocking Detachable Coils

    SciTech Connect

    Takahashi, Koji; Tanimura, Keiichi; Honda, Masanori; Kikuno, Motoyuki; Toei, Hisao; Hyodoh, Hideki; Furuse, Makoto; Yamada, Tomonori; Aburano, Tamio

    1999-05-15

    Purpose: To evaluate the indication and advantages of venous sac embolization of pulmonary arteriovenous malformations (PAVMs) using interlocking detachable coils (IDCs). Methods: We performed percutaneous embolization in 12 PAVMs in four patients using IDCs, initially placed in the venous sac or at the feeding artery to prevent systemic migration of additional coils. We placed the IDCs in the venous sac in PAVMs with the following vascular architecture: the draining vein was larger than the feeding arteries and both vessels were interposed with the venous sac or there were short feeding arteries. Results: Complete occlusion was achieved in all 12 PAVMs without significant complications. We deployed IDCs in the venous sac in eight PAVMs and in the feeding artery in four. Conclusion: Venous sac embolization may be beneficial in PAVMs with large out-flow vessels or short feeding arteries. IDCs are suitable for this procedure.

  3. The SAC51 Family Plays a Central Role in Thermospermine Responses in Arabidopsis.

    PubMed

    Cai, Qingqing; Fukushima, Hiroko; Yamamoto, Mai; Ishii, Nami; Sakamoto, Tomoaki; Kurata, Tetsuya; Motose, Hiroyasu; Takahashi, Taku

    2016-08-01

    The acaulis5 (acl5) mutant of Arabidopsis thaliana is defective in the biosynthesis of thermospermine and shows a dwarf phenotype associated with excess xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix protein. The sac51-d mutant has a premature termination codon in an upstream open reading frame (uORF) that is conserved among all four members of the SAC51 family, SAC51 and SACL1-SACL3 This suggests that thermospermine cancels the inhibitory effect of the uORF in main ORF translation. Another suppressor, sac57-d, has a mutation in the conserved uORF of SACL3 To define further the function of the SAC51 family in the thermospermine response, we analyzed T-DNA insertion mutants of each gene. Although sacl1-1 may not be a null allele, the quadruple mutant showed a semi-dwarf phenotype but with an increased level of thermospermine and decreased sensitivity to exogenous thermospermine that normally represses xylem differentiation. The sac51-1 sacl3-1 double mutant was also insensitive to thermospermine. These results suggest that SAC51 and SACL3 play a key role in thermospermine-dependent negative control of thermospermine biosynthesis and xylem differentiation. Using 5' leader-GUS (β-glucuronidase) fusion constructs, however, we detected a significant enhancement of the GUS activity by thermospermine only in SAC51 and SACL1 constructs. Furthermore, while acl5-1 sac51-1 showed the acl5 dwarf phenotype, acl5-1 sacl3-1 exhibited an extremely tiny-plant phenotype. These results suggest a complex regulatory network for the thermospermine response in which SAC51 and SACL3 function in parallel pathways. PMID:27388339

  4. Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

    PubMed Central

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias

    2007-01-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of

  5. Drug uptake into everted intestinal sacs. I. Enhancement by hypertonicity.

    PubMed

    Hurwitz, A; Gutman, Y

    1979-01-01

    The transfer of the cationic drugs, pralidoxime (PAM) and tetraethylammonium, and anionic ampicillin from the mucosal-to-serosal sides of everted rat jejunal sacs is enhanced by mucosal hypertonicity. PAM uptake, which is proportional to initial mucosal concentrations up to 2.3 mM, is enhanced by mucosal hypertonicity due to addition of sodium, potassium, lithium and choline chloride, sodium sulfate, and the nonionic solutes, urea, sucrose, and mannitol. Bicarbonate, Tris, or phosphate buffer and the presence of magnesium and calcium do not affect this hypertonicity-induced acceleration of PAM passage. Serosal osmolality has no effect on transfer and mucosal hypertonicity is equally effective in the presence and absence of a transmural osmotic gradient. This observation and minimal changes in the concentration of inulin placed in the sacs suggest that fluid shifts and solvent drag are not responsible for the enhanced mucosal-to-serosal transfer of PAM from hypertonic buffer. Mucosal hypertonicity at 450 mosmol/kg causes reversible enhancement of PAM transfer, whereas the effect of 600 mosmol/kg cannot be reversed by replacing the tissue in isotonic buffer. The effect of osmotic manipulation on PAM transfer across the intestine thus differs from its effect on the passage of other ionized species and drugs across other epithelia. PMID:434150

  6. Glucocorticoid and progesterone receptors in yolk sac placenta.

    PubMed

    Carbone, J P; Baldridge, R C; Magen, A B; Andrew, C L; Koszalka, T R; Brent, R L

    1986-01-01

    The parietal yolk sac (PYS) of the rat fetus at the 14th day of gestation contains glucocorticoid as well as progesterone receptors; both are present in the trophoblast cell layer. Following heat activation the receptors are capable of binding to deoxyribonucleic acid- (DNA-)cellulose. Glucocorticoid receptors, but not progesterone receptors, are also present in the visceral yolk sac (VYS) at the 14th day of gestation. Greater amounts (some 250 femtomoles/mg cytosol protein) of a glucocorticoid receptor are present in the VYS on the 17th day of gestation. The Kd is approximately 4 X 10(-9) M; following activation it also binds to DNA-cellulose. The elution pattern of the activated VYS receptor from diethylaminoethyl-(DEAE-)Sephadex, however, is similar to that found with kidney and colon rather than that of liver (i.e., it resembles corticosteroid binder IB rather than binder II) indicating a possible role in transport. Although the receptors are separate entities, progesterone competes as effectively as corticosterone for binding to the glucocorticoid receptors in both the PYS and and VYS, thus raising the question of the possible effect of changes in progesterone concentrations on the functioning of glucocorticoids during development.

  7. [Absorption of extractive Polygonum orientale in rat everted gut sacs].

    PubMed

    Liu, Yue; Tang, Li; Cao, Xu; Zheng, Lin; Wang, Ai-Min; Huang, Yong

    2014-06-01

    Using in vitro everted gut to investigate the intestinal absorption of the extracts from Polygonum orientale at different concentration. UPLC-MS/MS was used to detect the content of protocatechuic acid, isoorientin, orientin, vitexin, cynaroside, quercitrin, kaempferol-rhamnoside in different intestinal segments, then compared the results with the absorption of chemical components of extractive P. orientale in each intestinal segments, and calculated the absorption parameter. We took the statistic analysis with SPSS statistic software. The influence significance of each factors were analyzed to describe the character of absorption. The absorption of each component is linearity in different intestinal segments and different dose, and the square of coeficient correlation exceed 0.95, which consistent with zero order rate process. The K(a) increase along with the raised dosage of the extractive P. orientale (R2 > 0.95), indicated it is the passive absorption; different intestinal segments have different absorption. And the absorption trend in intestinal is duodenum, jejunum, ileum are greater than the colon. As ingredients are selectively absorbed in intestinal sac, the everted intestinal sac method is selected to assess the intestinal absorption charcteristics of ingredients of extractive P. orientale. PMID:25272855

  8. Angiotropic lymphoma occurring in a lacrimal sac oncocytoma.

    PubMed

    O'Connor, S R; Tan, J H-Y; Walewska, R; Brown, L J R; Lauder, I

    2002-10-01

    This report describes a case of angiotropic variant of diffuse large B cell lymphoma within a benign oncocytoma of the lacrimal sac. The occurrence of this rare lymphoma within a benign neoplasm has not been documented previously. An 87 year old woman presented with a swelling over the area of the left lacrimal sac, which histological examination revealed to be an oncocytoma. Many small blood vessels within the tumour were filled with large cytologically atypical cells, which stained positively for leucocyte common antigen and a B cell antigen, CD20, confirming the presence of a large B cell non-Hodgkin's lymphoma of angiotropic type. Angiotropic lymphoma is a very rare and usually highly aggressive variant of non-Hodgkin's lymphoma, which classically involves the central nervous system and skin, but has been described within most organs. Its occurrence within a benign neoplasm is probably coincidental, although a close association between oncocytic epithelium and normal lymphoid cells is recognised in Warthin's tumour of salivary and lacrimal glands.

  9. Potentiodynamic polarization effect on phase and microstructure of SAC305 solder in hydrochloric acid solution

    NASA Astrophysics Data System (ADS)

    Zaini, Nurwahida Binti Mohd; Nazeri, Muhammad Firdaus Bin Mohd

    2016-07-01

    The corrosion analysis of SAC305 lead free solder was investigated in Hydrochloric acid (HCl) solution. Potentiodynamic polarization was used to polarize the SAC305. The effect of polarization on the phase and microstructure were compared to as-prepared SAC305 solder. Potentiodynamic polarization introduces mixed corrosion products on the surface of SAC305 solder. The XRD analysis confirms that the mixed corrosion products emerged on the surface after polarization by formation of SnO and SnO2 of which confirmed that dissolution of Sn was dominant during polarization. Microstructure analysis reveal the presence of gap and porosities produced limits the protection offered by the passivation film.

  10. The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

    SciTech Connect

    Ikonomov, Ognian C. Filios, Catherine Sbrissa, Diego Chen, Xuequn Shisheva, Assia

    2013-10-18

    Highlights: •We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. •Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. •Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. •Phosphoinositide profiles are altered in breast cancer cells. •This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P{sub 2} synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P{sub 2} conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P{sub 2} in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or

  11. Adenylate cyclase in Arthrospira platensis responds to light through transcription.

    PubMed

    Kashith, M; Keerthana, B; Sriram, S; Ramamurthy, V

    2016-08-19

    Cyclic 3',5' adenosine monophosphate (cAMP) is a ubiquitous signaling molecule, but its role in higher plants was in doubt due to its very low concentration. In this study we wanted to look at the flux of cAMP in response to light in algae, considered to be the more primitive form of photosynthetic organisms. While it did not fluctuate very much in the tested green algae, in the cyanobacterium Arthrospira platensis its level was closely linked to exposure to light. The expression from cyaC, the major isoform of adenylate cyclase was strongly influenced by exposure of the cells to light. There was about 300 fold enhancement of cyaC transcripts in cells exposed to light compared to the transcripts in cells in the dark. Although post-translational regulation of adenylate cyclase activity has been widely known, our studies suggest that transcriptional control could also be an important aspect of its regulation in A. platensis. PMID:27311855

  12. Structural analysis of an oxygen-regulated diguanylate cyclase.

    PubMed

    Tarnawski, Miroslaw; Barends, Thomas R M; Schlichting, Ilme

    2015-11-01

    Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

  13. Clearance of chlamydial elementary bodies from the conjunctival sac

    SciTech Connect

    Taylor, H.R.; Velez, V.L.

    1987-07-01

    The rate of disappearance of inactivated Chlamydia trachomatis elementary body (EB) preparations from the conjunctival sac was studied in monkeys. Direct fluorescent antibody (DFA) cytology showed that the majority of EB had been cleared from the eye within 24 hr of the inoculation of 1 X 10(6) inactivated EB, although small numbers of EB could be detected for up to 144 hr. The rate of clearance in normal and ocular immune animals did not differ, and formalin-killed and UV-inactivated EBs disappeared at a comparable rate. These studies suggest that chlamydial EB are cleared relatively quickly from the eye and support the notion that EBs detected by DFA cytology indicate the presence of current infection.

  14. Endometriosis within a left-sided inguinal hernia sac

    PubMed Central

    Albutt, Katherine; Glass, Charity; Odom, Stephen; Gupta, Alok

    2014-01-01

    Endometriosis is a common gynecologic disorder wherein ectopic endometrial glands and stroma are found at extrauterine sites. Extrapelvic endometriosis is a well-documented, yet rare, disease entity that can affect almost any organ system. Inguinal endometriosis is an extremely rare disease entity characterized by tender inguinal swelling. Here we report a case of a sudden-onset and acutely painful left inguinal hernia with concordant endometriosis. A review of the literature is presented. The presence of isolated endometriosis contained within a left-sided inguinal hernia sac has, to our knowledge, never been reported. Often diagnosed incidentally or on histologic examination, general surgeons should consider inguinal endometriosis in the differential diagnosis of inguinal masses, even in the absence of catamenial symptoms. Surgical excision, with gynecologic follow-up, is locally curative and the treatment of choice for inguinal endometriosis. PMID:24876515

  15. Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

    PubMed

    Koshimizu, Taka-aki; Tsuchiya, Hiroyoshi; Tsuda, Hidetoshi; Fujiwara, Yoko; Shibata, Katsushi; Hirasawa, Akira; Tsujimoto, Gozoh; Fujimura, Akio

    2010-02-19

    Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

  16. The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

    PubMed Central

    Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

    1972-01-01

    Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

  17. The influence of locomotion on air-sac pressures in little penguins.

    PubMed

    Boggs, D F; Baudinette, R V; Frappell, P B; Butler, P J

    2001-10-01

    Air-sac pressures have been reported to oscillate with wing beat in flying magpies and with foot paddling in diving ducks. We sought to determine the impact on air-sac pressure of wing beats during swimming and of the step cycle during walking in little penguins (Eudyptula minor). Fluctuations averaged 0.16+/-0.06 kPa in the interclavicular air sacs, but only 0.06+/-0.04 kPa in the posterior thoracic sac, generating a small differential pressure between sacs of 0.06+/-0.02 kPa (means +/- S.E.M., N=4). These fluctuations occurred at approximately 3 Hz and corresponded to wing beats during swimming, indicated by electromyograms from the pectoralis and supracoracoideus muscles. There was no abdominal muscle activity associated with swimming or exhalation, but the abdominal muscles were active with the step cycle in walking penguins, and oscillations in posterior air-sac pressure (0.08+/-0.038 kPa) occurred with steps. We conclude that high-frequency oscillations in differential air-sac pressure enhance access to and utilization of the O(2) stores in the air sacs during a dive. PMID:11707507

  18. Diagnostic imaging features of normal anal sacs in dogs and cats.

    PubMed

    Jung, Yechan; Jeong, Eunseok; Park, Sangjun; Jeong, Jimo; Choi, Ul Soo; Kim, Min-Su; Kim, Namsoo; Lee, Kichang

    2016-09-30

    This study was conducted to provide normal reference features for canine and feline anal sacs using ultrasound, low-field magnetic resonance imaging (MRI) and radiograph contrast as diagnostic imaging tools. A total of ten clinically normal beagle dogs and eight clinically normally cats were included. General radiography with contrast, ultrasonography and low-field MRI scans were performed. The visualization of anal sacs, which are located at distinct sites in dogs and cats, is possible with a contrast study on radiography. Most surfaces of the anal sacs tissue, occasionally appearing as a hyperechoic thin line, were surrounded by the hypoechoic external sphincter muscle on ultrasonography. The normal anal sac contents of dogs and cats had variable echogenicity. Signals of anal sac contents on low-field MRI varied in cats and dogs, and contrast medium using T1-weighted images enhanced the anal sac walls more obviously than that on ultrasonography. In conclusion, this study provides the normal features of anal sacs from dogs and cats on diagnostic imaging. Further studies including anal sac evaluation are expected to investigate disease conditions.

  19. Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2014-04-01

    Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS.

  20. Finite element analysis of stresses developed in the blood sac of a left ventricular assist device.

    PubMed

    Haut Donahue, T L; Dehlin, W; Gillespie, J; Weiss, W J; Rosenberg, G

    2009-05-01

    The goal of this research is to develop a 3D finite element (FE) model of a left ventricular assist device (LVAD) to predict stresses in the blood sac. The hyperelastic stress-strain curves for the segmented poly(ether polyurethane urea) (SPEUU) blood sac were determined in both tension and compression using a servo-hydraulic testing system at various strain rates. Over the range of strain rates studied, the sac was not strain rate sensitive, however the material response was different for tension versus compression. The experimental tension and compression properties were used in a FE model that consisted of the pusher plate, blood sac and pump case. A quasi-static analysis was used to allow for nonlinearities due to contact and material deformation. The 3D FE model showed that blood sac stresses are not adversely affected by the location of the inlet and outlet ports of the device and that over the systolic ejection phase of the simulation the prediction of blood sac stresses from the full 3D model and an axisymmetric model are the same. Minimizing stresses in the blood sac will increase the longevity of the blood sac in vivo. PMID:19131267

  1. 74. SAC control center addition sect 9top three floors, first ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    74. SAC control center addition sect 9-top three floors, first floor plan, drawing number AS-BLT.AW30-02-03, dated May, 1958 - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Headquarters Building, 901 SAC Boulevard, Bellevue, Sarpy County, NE

  2. Mammalian laryngseal air sacs add variability to the vocal tract impedance: Physical and computational modeling

    PubMed Central

    Riede, Tobias; Tokuda, Isao T.; Munger, Jacob B.; Thomson, Scott L.

    2008-01-01

    Cavities branching off the main vocal tract are ubiquitous in nonhumans. Mammalian air sacs exist in human relatives, including all four great apes, but only a substantially reduced version exists in humans. The present paper focuses on acoustical functions of the air sacs. The hypotheses are investigated on whether the air sacs affect amplitude of utterances and∕or position of formants. A multilayer synthetic model of the vocal folds coupled with a vocal tract model was utilized. As an air sac model, four configurations were considered: open and closed uniform tube-like side branches, a rigid cavity, and an inflatable cavity. Results suggest that some air sac configurations can enhance the sound level. Furthermore, an air sac model introduces one or more additional resonance frequencies, shifting formants of the main vocal tract to some extent but not as strongly as previously suggested. In addition, dynamic range of vocalization can be extended by the air sacs. A new finding is also an increased variability of the vocal tract impedance, leading to strong nonlinear source-filter interaction effects. The experiments demonstrated that air-sac-like structures can destabilize the sound source. The results were validated by a transmission line computational model. PMID:18647005

  3. Diagnostic imaging features of normal anal sacs in dogs and cats

    PubMed Central

    Jung, Yechan; Jeong, Eunseok; Park, Sangjun; Jeong, Jimo; Choi, Ul Soo; Kim, Min-Su; Kim, Namsoo

    2016-01-01

    This study was conducted to provide normal reference features for canine and feline anal sacs using ultrasound, low-field magnetic resonance imaging (MRI) and radiograph contrast as diagnostic imaging tools. A total of ten clinically normal beagle dogs and eight clinically normally cats were included. General radiography with contrast, ultrasonography and low-field MRI scans were performed. The visualization of anal sacs, which are located at distinct sites in dogs and cats, is possible with a contrast study on radiography. Most surfaces of the anal sacs tissue, occasionally appearing as a hyperechoic thin line, were surrounded by the hypoechoic external sphincter muscle on ultrasonography. The normal anal sac contents of dogs and cats had variable echogenicity. Signals of anal sac contents on low-field MRI varied in cats and dogs, and contrast medium using T1-weighted images enhanced the anal sac walls more obviously than that on ultrasonography. In conclusion, this study provides the normal features of anal sacs from dogs and cats on diagnostic imaging. Further studies including anal sac evaluation are expected to investigate disease conditions. PMID:26645338

  4. Ground water in the Cul-de-Sac Plain, Haiti

    USGS Publications Warehouse

    Taylor, George C.; Lemoine, Rémy C.

    1949-01-01

    The Cul-de-Sac Plain is perhaps the most important agricultural area in Haiti because of its nearness and accessibility to Port-au-Prince, the nation's capital, metropolis, and principal seaport. Most of the agricultural produce consumed in Port-au-Prince as well as a considerable part of that exported from Haiti is grown in the plain. Because of variable and poorly distributed rainfall, high temperature, and high evaporation, semiarid climatic conditions prevail in the plain. Irrigation is, therefore, necessary for successful farming. There are no regulatory or storage facilities on the streams that enter the plain, but the mean and low-water stream flow and the discharge of springs are almost entirely appropriated for irrigation. Ground water has been utilized for irrigation to an increasing extent by the Haitian American Sugar Company, which has put down about 100 wells in the plain since 1919. Outside the existing irrigated areas of the plain are large tracts of potentially irrigable land that are uncultivated and agriculturally unproductive for lack of water. The object of the present study was to determine the possibilities of bringing these lands into cultivation by irrigation from wells. This study was part of a larger program of the Food Supply Division, Institute of Inter-American Affairs, to increase the production of food in Haiti. From September through November 1948 the senior author, a member of the U. S. Geological Survey, spent three months in the field in an investigation of the geology and ground-water resources of the Cul-de-Sac Plain. He was ably assisted by Mr. Rémy C. Lemoine, Haitian engineer-geologist, employed by the Food Supply Division. The field work included principally the geologic mapping of' the plain and the adjacent mountain borders, a ground-water inventory of existing wells and springs, and a general evaluation of significant geologic and hydrologic features.

  5. Ground water in the Cul-de-Sac Plain, Haiti

    USGS Publications Warehouse

    Taylor, George C.; Lemoine, Rémy C.

    1949-01-01

    The Cul-de-Sac Plain is perhaps the most important agricultural area in Haiti because of its nearness and accessibility to Port-au-Prince, the nation's capital, metropolis, and principal seaport. Most of the agricultural produce consumed in Port-au-Prince as well as a considerable part of that exported from Haiti is grown in the plain.Because of variable and poorly distributed rainfall, high temperature, and high evaporation, semiarid climatic conditions prevail in the plain. Irrigation is, therefore, necessary for successful farming. There are no regulatory or storage facilities on the streams that enter the plain, but the mean and low-water stream flow and the discharge of springs are almost entirely appropriated for irrigation. Ground water has been utilized for irrigation to an increasing extent by the Haitian American Sugar Company, which has put down about 100 wells in the plain since 1919.Outside the existing irrigated areas of the plain are large tracts of potentially irrigable land that are uncultivated and agriculturally unproductive for lack of water. The object of the present study was to determine the possibilities of bringing these lands into cultivation by irrigation from wells. This study was part of a larger program of the Food Supply Division, Institute of Inter-American Affairs, to increase the production of food in Haiti.From September through November 1948 the senior author, a member of the U. S. Geological Survey, spent three months in the field in an investigation of the geology and ground-water resources of the Cul-de-Sac Plain. He was ably assisted by Mr. Rémy C. Lemoine, Haitian engineer-geologist, employed by the Food Supply Division. The field work included principally the geologic mapping of' the plain and the adjacent mountain borders, a ground-water inventory of existing wells and springs, and a general evaluation of significant geologic and hydrologic features.

  6. Development of the endolymphatic sac in chick embryos, with reference to the degradation of otoconia

    NASA Technical Reports Server (NTRS)

    Yoshihara, T.; Kaname, H.; Narita, N.; Ishii, T.; Igarashi, M.; Fermin, C. D.

    1992-01-01

    The endolymphatic sac of chick embryos (from embryonic day 7 to 1-day-old chicks) was studied light- and electron-microscopically. At stage 30-31 (embryonic day 7-7.5), the epithelial cells of the endolymphatic sac were cuboidal to columnar in shape. Microvilli were relatively well developed. The intercellular space was wide. In the endolymphatic space of the endolymphatic sac, varying shapes and sizes of otoconia-like bodies were often observed. Intracytoplasmic phagosomes containing these bodies were rarely found. After stage 37 (embryonic day 11), otoconia-like bodies in the endolymphatic sac decreased in number and size. They were almost the same as the otoconia in the macular organs, ultrastructurally. These findings indicate that the endolymphatic sac of the chick embryos may possess the function of otoconial degradation and removal of calcium from otoconia.

  7. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory. PMID:27695398

  8. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.

  9. Soluble guanylate cyclase as a novel treatment target for osteoporosis.

    PubMed

    Joshua, Jisha; Schwaerzer, Gerburg K; Kalyanaraman, Hema; Cory, Esther; Sah, Robert L; Li, Mofei; Vaida, Florin; Boss, Gerry R; Pilz, Renate B

    2014-12-01

    Osteoporosis is a major health problem leading to fractures that cause substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, creating a need for novel bone-anabolic agents. We previously showed that the nitric oxide/cyclic GMP (cGMP)/protein kinase G pathway mediates some of the anabolic effects of estrogens and mechanical stimulation in osteoblasts and osteocytes, leading us to hypothesize that cGMP-elevating agents may have bone-protective effects. We tested cinaciguat, a prototype of a novel class of soluble guanylate cyclase activators, in a mouse model of estrogen deficiency-induced osteoporosis. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat or low-dose 17β-estradiol. Microcomputed tomography of tibiae showed that cinaciguat significantly improved trabecular bone microarchitecture in ovariectomized animals, with effect sizes similar to those obtained with estrogen replacement therapy. Cinaciguat reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Compared with 17β-estradiol, which completely reversed the ovariectomy-induced increase in osteoclast number, cinaciguat had little effect on osteoclasts. Direct guanylate cyclase stimulators have been extremely well tolerated in clinical trials of cardiovascular diseases, and our findings provide proof-of-concept for this new class of drugs as a novel, anabolic treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone. PMID:25188528

  10. Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation.

    PubMed

    Schmidt, Kurt; Neubauer, Andrea; Kolesnik, Bernd; Stasch, Johannes-Peter; Werner, Ernst R; Gorren, Antonius C F; Mayer, Bernd

    2012-09-01

    Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress. PMID:22648973

  11. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  12. Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes*

    PubMed Central

    Rosenberg, Jonathan; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M.

    2015-01-01

    The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement. PMID:25605729

  13. Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

    PubMed

    Hungen, K V; Roberts, S; Hill, D F

    1975-08-22

    Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

  14. Nordic rattle: the hoarse vocalization and the inflatable laryngeal air sac of reindeer (Rangifer tarandus).

    PubMed

    Frey, Roland; Gebler, Alban; Fritsch, Guido; Nygrén, Kaarlo; Weissengruber, Gerald E

    2007-02-01

    Laryngeal air sacs have evolved convergently in diverse mammalian lineages including insectivores, bats, rodents, pinnipeds, ungulates and primates, but their precise function has remained elusive. Among cervids, the vocal tract of reindeer has evolved an unpaired inflatable ventrorostral laryngeal air sac. This air sac is not present at birth but emerges during ontogenetic development. It protrudes from the laryngeal vestibulum via a short duct between the epiglottis and the thyroid cartilage. In the female the growth of the air sac stops at the age of 2-3 years, whereas in males it continues to grow up to the age of about 6 years, leading to a pronounced sexual dimorphism of the air sac. In adult females it is of moderate size (about 100 cm3), whereas in adult males it is large (3000-4000 cm3) and becomes asymmetric extending either to the left or to the right side of the neck. In both adult females and males the ventral air sac walls touch the integument. In the adult male the air sac is laterally covered by the mandibular portion of the sternocephalic muscle and the skin. Both sexes of reindeer have a double stylohyoid muscle and a thyroepiglottic muscle. Possibly these muscles assist in inflation of the air sac. Head-and-neck specimens were subjected to macroscopic anatomical dissection, computer tomographic analysis and skeletonization. In addition, isolated larynges were studied for comparison. Acoustic recordings were made during an autumn round-up of semi-domestic reindeer in Finland and in a small zoo herd. Male reindeer adopt a specific posture when emitting their serial hoarse rutting calls. Head and neck are kept low and the throat region is extended. In the ventral neck region, roughly corresponding to the position of the large air sac, there is a mane of longer hairs. Neck swelling and mane spreading during vocalization may act as an optical signal to other males and females. The air sac, as a side branch of the vocal tract, can be considered as an

  15. Nordic rattle: the hoarse vocalization and the inflatable laryngeal air sac of reindeer (Rangifer tarandus)

    PubMed Central

    Frey, Roland; Gebler, Alban; Fritsch, Guido; Nygrén, Kaarlo; Weissengruber, Gerald E

    2007-01-01

    Laryngeal air sacs have evolved convergently in diverse mammalian lineages including insectivores, bats, rodents, pinnipeds, ungulates and primates, but their precise function has remained elusive. Among cervids, the vocal tract of reindeer has evolved an unpaired inflatable ventrorostral laryngeal air sac. This air sac is not present at birth but emerges during ontogenetic development. It protrudes from the laryngeal vestibulum via a short duct between the epiglottis and the thyroid cartilage. In the female the growth of the air sac stops at the age of 2–3 years, whereas in males it continues to grow up to the age of about 6 years, leading to a pronounced sexual dimorphism of the air sac. In adult females it is of moderate size (about 100 cm3), whereas in adult males it is large (3000–4000 cm3) and becomes asymmetric extending either to the left or to the right side of the neck. In both adult females and males the ventral air sac walls touch the integument. In the adult male the air sac is laterally covered by the mandibular portion of the sternocephalic muscle and the skin. Both sexes of reindeer have a double stylohyoid muscle and a thyroepiglottic muscle. Possibly these muscles assist in inflation of the air sac. Head-and-neck specimens were subjected to macroscopic anatomical dissection, computer tomographic analysis and skeletonization. In addition, isolated larynges were studied for comparison. Acoustic recordings were made during an autumn round-up of semi-domestic reindeer in Finland and in a small zoo herd. Male reindeer adopt a specific posture when emitting their serial hoarse rutting calls. Head and neck are kept low and the throat region is extended. In the ventral neck region, roughly corresponding to the position of the large air sac, there is a mane of longer hairs. Neck swelling and mane spreading during vocalization may act as an optical signal to other males and females. The air sac, as a side branch of the vocal tract, can be considered as

  16. Conjunctival sac bacterial flora isolated prior to cataract surgery

    PubMed Central

    Suto, Chikako; Morinaga, Masahiro; Yagi, Tomoko; Tsuji, Chieko; Toshida, Hiroshi

    2012-01-01

    Objective To determine the trends of conjunctival sac bacterial flora isolated from patients prior to cataract surgery. Subjects and methods The study comprised 579 patients (579 eyes) who underwent cataract surgery. Specimens were collected by lightly rubbing the inferior palpebral conjunctival sac with a sterile cotton swab 2 weeks before surgery, and then cultured for isolation of bacteria and antimicrobial sensitivity testing. The bacterial isolates and percentage of drug-resistant isolates were compared among age groups and according to whether or not patients had diabetes mellitus, hyperlipidemia, dialysis therapy, oral steroid use, dry eye syndrome, or allergic conjunctivitis. Results The bacterial isolation rate was 39.2%. There were 191 strains of Gram-positive cocci, accounting for the majority of all isolates (67.0%), among which methicillin-sensitive coagulase-negative staphylococci was the most frequent (127 strains, 44.5%), followed by methicillin-resistant coagulase-negative staphylococci (37 strains, 12.7%). All 76 Gram-positive bacillary isolates (26.7%) were from the genus Corynebacterium. Among the 16 Gram-negative bacillary isolates (5.9%), the most frequent was Escherichia coli (1.0%). The bacterial isolation rate was higher in patients >60 years old, and was lower in patients with dry eye syndrome, patients under topical treatment for other ocular disorders, and patients with hyperlipidemia. There was no significant difference in bacterial isolation rate with respect to the presence/absence of diabetes mellitus, steroid therapy, dialysis, or a history of allergic conjunctivitis. Methicillin-resistant coagulase-negative staphylococci showed a significantly higher detection rate in diabetic patients than nondiabetic patients (20.3% versus 7.0%, P < 0.05). The percentage of all isolates resistant to levofloxacin, cefmenoxime, and tobramycin was 14.0%, 15.2%, and 17.9%, respectively, with no significant differences among these drugs. Conclusion

  17. The First Transmembrane Domain of Lipid Phosphatase SAC1 Promotes Golgi Localization

    PubMed Central

    Wang, Jinzhi; Chen, Juxing; Enns, Caroline A.; Mayinger, Peter

    2013-01-01

    The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region. PMID:23936490

  18. The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

    PubMed Central

    Liu, Yang; Boukhelifa, Malika; Tribble, Emily; Morin-Kensicki, Elizabeth; Uetrecht, Andrea; Bear, James E.

    2008-01-01

    Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties. PMID:18480408

  19. The Sac1 phosphoinositide phosphatase regulates Golgi membrane morphology and mitotic spindle organization in mammals.

    PubMed

    Liu, Yang; Boukhelifa, Malika; Tribble, Emily; Morin-Kensicki, Elizabeth; Uetrecht, Andrea; Bear, James E; Bankaitis, Vytas A

    2008-07-01

    Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties.

  20. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    PubMed Central

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  1. Crystal Structures of the Catalytic Domain of Human Soluble Guanylate Cyclase

    PubMed Central

    Allerston, Charles K.; von Delft, Frank; Gileadi, Opher

    2013-01-01

    Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain. PMID:23505436

  2. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that

  3. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.

  4. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin. PMID:22642150

  5. Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

    2015-08-01

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).

  6. SAC phosphoinositide phosphatases at the tonoplast mediate vacuolar function in Arabidopsis

    PubMed Central

    Nováková, Petra; Hirsch, Sibylle; Feraru, Elena; Tejos, Ricardo; van Wijk, Ringo; Viaene, Tom; Heilmann, Mareike; Lerche, Jennifer; De Rycke, Riet; Feraru, Mugurel I.; Grones, Peter; Van Montagu, Marc; Heilmann, Ingo; Munnik, Teun; Friml, Jiří

    2014-01-01

    Phosphatidylinositol (PtdIns) is a structural phospholipid that can be phosphorylated into various lipid signaling molecules, designated polyphosphoinositides (PPIs). The reversible phosphorylation of PPIs on the 3, 4, or 5 position of inositol is performed by a set of organelle-specific kinases and phosphatases, and the characteristic head groups make these molecules ideal for regulating biological processes in time and space. In yeast and mammals, PtdIns3P and PtdIns(3,5)P2 play crucial roles in trafficking toward the lytic compartments, whereas the role in plants is not yet fully understood. Here we identified the role of a land plant-specific subgroup of PPI phosphatases, the suppressor of actin 2 (SAC2) to SAC5, during vacuolar trafficking and morphogenesis in Arabidopsis thaliana. SAC2–SAC5 localize to the tonoplast along with PtdIns3P, the presumable product of their activity. In SAC gain- and loss-of-function mutants, the levels of PtdIns monophosphates and bisphosphates were changed, with opposite effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles was defective. Moreover, multiple sac knockout mutants had an increased number of smaller storage and lytic vacuoles, whereas extralarge vacuoles were observed in the overexpression lines, correlating with various growth and developmental defects. The fragmented vacuolar phenotype of sac mutants could be mimicked by treating wild-type seedlings with PtdIns(3,5)P2, corroborating that this PPI is important for vacuole morphology. Taken together, these results provide evidence that PPIs, together with their metabolic enzymes SAC2–SAC5, are crucial for vacuolar trafficking and for vacuolar morphology and function in plants. PMID:24550313

  7. The effect of pigeon yolk sac fluid on the growth behavior of calcium carbonate crystals.

    PubMed

    Song, Juan; Cheng, Haixia; Shen, Xinyu; Tong, Hua

    2015-03-01

    Previous experiments have proved that thermodynamically unstable calcium carbonate vaterite can exist for long periods in the yolk sac of a pigeon embryo. The aim of this article was to demonstrate the effect of in vitro mineralization of yolk sac fluid on calcium carbonate by direct precipitation. Experiments were conducted using pigeon yolk sac fluid and using lecithin extracted from pigeon yolk sac fluid as a control to investigate the regulating effects of the organic components in the embryo on the formation of the calcium carbonate precipitate. Multiple characterization methods were employed to study the various morphological patterns, sizes, crystal growth, and crystal phase transformations of the calcium carbonate precipitates as regulated by the yolk sac fluid extracted at different stages of incubation. The experimental results demonstrate that as the incubation proceeds towards the later stages, the composition and environmental features of the yolk sac fluid become more favorable for the formation of relatively unstable calcium carbonate phases with high energies of the vaterite state. The experiments conducted with extracted lecithin as the template for crystal growth yielded similar results. A large amount of organic molecules with polar functional groups carried by the yolk sac fluid have strong effects and can both initially induce the crystallization and regulate the aggregation of calcium carbonate. Furthermore, this regulation process is found to be closely related to the lecithin contained in yolk sac fluid. These observations confirm the changes in yolk sac fluid composition during incubation have significant effects on the production of vaterite, which implicates the calcium transport during embryo growth.

  8. XPS of oxygen atoms on Ag(111) and Ag(110) surfaces: accurate study with SAC/SAC-CI combined with dipped adcluster model.

    PubMed

    Ishikawa, Atsushi; Nakatsuji, Hiroshi

    2013-08-01

    O1s core-electron binding energies (CEBE) of the atomic oxygens on different Ag surfaces were investigated by the symmetry adapted cluster-configuration interaction (SAC-CI) method combined with the dipped adcluster model, in which the electron exchange between bulk metal and adsorbate is taken into account properly. Electrophilic and nucleophilic oxygens (O(elec) and O(nuc)) that might be important for olefin epoxidation in a low-oxygen coverage condition were focused here. We consider the O1s CEBE as a key property to distinguish the surface oxygen states, and series of calculation was carried out by the Hartree-Fock, Density functional theory, and SAC/SAC-CI methods. The experimental information and our SAC/SAC-CI results indicate that O(elec) is the atomic oxygen adsorbed on the fcc site of Ag(111) and that O(nuc) is the one on the reconstructed added-row site of Ag(110) and that one- and two-electron transfers occur, respectively, to the O(elec) and O(nuc) adclusters from the silver surface.

  9. Primary yolk sac tumor of the gluteus: a case report and literature review

    PubMed Central

    Li, Bo; Jiang, Qianqian; Zhang, Shitai; Zhou, Yang; Zhang, Qing-Fu; OuYang, Ling

    2016-01-01

    Yolk sac tumor (YST) is a common malignant primitive germ cell tumor that often exhibits differentiation into endodermal structures. They most commonly occur in childhood and adolescence and are rare after the age of 40 years. Derived from the yolk sac during the embryonic period, YSTs can occur in the gonads and germ cells because the tumor cells migrate from the yolk sac toward the gonads. Here, we present a rare case of primary gluteus YST in a 3-year-old girl. She received BEP chemotherapy (bleomycin + etoposide + cisplatin) after surgical resection. There was no evidence of recurrence 7 months after primary treatment. PMID:27536133

  10. Primary yolk sac tumor of the gluteus: a case report and literature review.

    PubMed

    Li, Bo; Jiang, Qianqian; Zhang, Shitai; Zhou, Yang; Zhang, Qing-Fu; OuYang, Ling

    2016-01-01

    Yolk sac tumor (YST) is a common malignant primitive germ cell tumor that often exhibits differentiation into endodermal structures. They most commonly occur in childhood and adolescence and are rare after the age of 40 years. Derived from the yolk sac during the embryonic period, YSTs can occur in the gonads and germ cells because the tumor cells migrate from the yolk sac toward the gonads. Here, we present a rare case of primary gluteus YST in a 3-year-old girl. She received BEP chemotherapy (bleomycin + etoposide + cisplatin) after surgical resection. There was no evidence of recurrence 7 months after primary treatment. PMID:27536133

  11. Surveillance Analysis Computer System (SACS): Software requirements specification (SRS). Revision 2

    SciTech Connect

    Glasscock, J.A.

    1995-03-08

    This document is the primary document establishing requirements for the Surveillance Analysis Computer System (SACS) database, an Impact Level 3Q system. SACS stores information on tank temperatures, surface levels, and interstitial liquid levels. This information is retrieved by the customer through a PC-based interface and is then available to a number of other software tools. The software requirements specification (SRS) describes the system requirements for the SACS Project, and follows the Standard Engineering Practices (WHC-CM-6-1), Software Practices (WHC-CM-3-10) and Quality Assurance (WHC-CM-4-2, QR 19.0) policies.

  12. Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

    ERIC Educational Resources Information Center

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

  13. Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity.

    PubMed

    Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K; Makino, Clint L

    2015-04-24

    By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca(2+)]i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED50 of 27 mM for ROS-GC1 and 39 mM for ROS-GC2. The effect required neither Ca(2+) nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca(2+)]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity. PMID:25767116

  14. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  15. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  16. Guanylyl cyclase C signaling axis and colon cancer prevention.

    PubMed

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-09-28

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  17. Murine Guanylate Cyclase C Regulates Colonic Injury and Inflammation1

    PubMed Central

    Steinbrecher, Kris A.; Harmel-Laws, Eleana; Garin-Laflam, Monica P.; Mann, Elizabeth A.; Bezerra, Lucas D.; Hogan, Simon P.; Cohen, Mitchell B.

    2011-01-01

    Guanylate cyclase C (GUCY2C or GC-C) and its ligands, guanylin (GUCA2A or Gn) and uroguanylin (GUCA2B or Ugn), are expressed in intestinal epithelial cells (IECs) and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine if GC-C and its ligands direct the course of intestinal inflammation. Here, we show that DSS-induced clinical disease and histological damage to the colonic mucosa were significantly less severe in GC-C−/− mice and moderately reduced in Gn−/− animals. Relative to wildtype controls, GC-C−/− and Gn−/− mice had reduced apoptosis and increased proliferation of IECs during DSS colitis. Basal and DSS-induced production of resistin-like molecule β (RELMβ) was substantially diminished in GC-C−/− mice. RELMβ is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNFα and IFNγ production was minimal in GC-C−/− animals. RELMβ and cytokine levels were similar to wildtype in Gn−/− mice, however. Colonic instillation of recombinant RELMβ by enema into GC-C−/− mice restores sensitivity to DSS-mediated mucosal injury. These findings demonstrate a novel role for GC-C signaling in facilitating mucosal wounding and inflammation and further suggest that this may be mediated, in part, through control of RELMβ production. PMID:21555532

  18. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  19. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

  20. Histological and histochemical analyses of the cuttlebone sac of the golden cuttlefish Sepia esculenta

    NASA Astrophysics Data System (ADS)

    Zheng, Xiaodong; Xiao, Shu; Wang, Zhaoping; Wang, Rucai

    2007-10-01

    The secretion function of mantle is closely related to shell formation in some bivalves and gastropods. Up to now, few researches have been reported for cuttlebone formation in the class Cephalopoda. In this study, the structure and secretion function of cuttlebone sac of the golden cuttlefish Sepia esculenta was analyzed using the histological and histochemical methods. The results showed that high and columnar cells located in sac epithelium, and flat cells existed near the base membrane. A lot of fibroblasts were found in the lateral mantle collective tissue. Some mucus, mucopolysaccharide and alkaline phosphatase (ALP) were found in the sac. The ultrastructural characteristics of Quasi-connective-tissue-calcium cells (QCTCC) were observed using a transmission electron microscope (TEM). The relationship between cuttlebone sac secretion function and shell formation was discussed.

  1. Evidence for Avian Intrathoracic Air Sacs in a New Predatory Dinosaur from Argentina

    PubMed Central

    Sereno, Paul C.; Martinez, Ricardo N.; Wilson, Jeffrey A.; Varricchio, David J.; Alcober, Oscar A.; Larsson, Hans C. E.

    2008-01-01

    Background Living birds possess a unique heterogeneous pulmonary system composed of a rigid, dorsally-anchored lung and several compliant air sacs that operate as bellows, driving inspired air through the lung. Evidence from the fossil record for the origin and evolution of this system is extremely limited, because lungs do not fossilize and because the bellow-like air sacs in living birds only rarely penetrate (pneumatize) skeletal bone and thus leave a record of their presence. Methodology/Principal Findings We describe a new predatory dinosaur from Upper Cretaceous rocks in Argentina, Aerosteon riocoloradensis gen. et sp. nov., that exhibits extreme pneumatization of skeletal bone, including pneumatic hollowing of the furcula and ilium. In living birds, these two bones are pneumatized by diverticulae of air sacs (clavicular, abdominal) that are involved in pulmonary ventilation. We also describe several pneumatized gastralia (“stomach ribs”), which suggest that diverticulae of the air sac system were present in surface tissues of the thorax. Conclusions/Significance We present a four-phase model for the evolution of avian air sacs and costosternal-driven lung ventilation based on the known fossil record of theropod dinosaurs and osteological correlates in extant birds: (1) Phase I—Elaboration of paraxial cervical air sacs in basal theropods no later than the earliest Late Triassic. (2) Phase II—Differentiation of avian ventilatory air sacs, including both cranial (clavicular air sac) and caudal (abdominal air sac) divisions, in basal tetanurans during the Jurassic. A heterogeneous respiratory tract with compliant air sacs, in turn, suggests the presence of rigid, dorsally attached lungs with flow-through ventilation. (3) Phase III—Evolution of a primitive costosternal pump in maniraptoriform theropods before the close of the Jurassic. (4) Phase IV—Evolution of an advanced costosternal pump in maniraptoran theropods before the close of the

  2. Deposition of anal-sac secretions by captive wolves (Canis lupus)

    USGS Publications Warehouse

    Asa, C.S.; Peterson, E.K.; Seal, U.S.; Mech, L.D.

    1985-01-01

    Deposition of anal-sac secretions by captive wolves was investigated by a labelling technique using protein-bound iodine125 and food dye. Wolves deposited secretions on some but not all scats. Adult males, especially the alpha male, deposited anal-sac secretions more frequently while defecating than did females or juveniles. Secretions sometimes also were deposited independently of defecation, suggesting a dual role in communication by these substances.

  3. Alterations in adipocyte adenylate cyclase activity in morbidly obese and formerly morbidly obese humans.

    PubMed

    Martin, L F; Klim, C M; Vannucci, S J; Dixon, L B; Landis, J R; LaNoue, K F

    1990-08-01

    Studies examining animal models of genetic obesity have identified defects in adipocyte hormone-stimulated lipolysis that involve the adenylate cyclase transmembrane signaling system, specifically those components that decrease adenylate cyclase activity. To determine whether obese people demonstrate alterations in adenylate cyclase activity that could contribute to the maintenance of obesity by inhibiting lipolysis, we examined human adipocytes from patients who were lean, obese, or formerly obese. Fat samples were obtained from the lower abdomen of 14 women who were morbidly obese (obese group), from 10 women who were formerly morbidly obese and had lost weight after gastric stapling (postobese group), and from 10 similarly aged women of normal weight (controls). Adipocyte adenylate cyclase activity was determined under ligand-free (no stimulatory or inhibitory influences present), hormone-stimulated (isoproterenol, 10(-6) mmol/L), and maximal (cells stimulated with 10 mumol/L forskolin) conditions by measuring cyclic adenosine monophosphate (cAMP) levels by radioimmunoassay. The activity of adenylate cyclase was significantly different (p less than 0.01) in the three groups. Adipocytes from obese women had lower levels of cyclase activity under both ligand-free (5% vs 16% of maximal) and hormone-stimulated conditions (76% vs 100% of maximal) than adipocytes from normal women. Postobese women had levels of hormone-stimulated cAMP identical to those of normal women but still had abnormal ligand-free levels (under 5%). These results suggest the presence of an alteration in adipocyte adenylate cyclase regulation in morbidly obese women that is not entirely corrected when weight is lost after food intake is reduced by gastric stapling. This alteration in ligand-free cAMP activity may contribute to the development and maintenance of obesity. PMID:2166354

  4. Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

    SciTech Connect

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M.; Burd, Christopher G.

    2014-08-25

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  5. A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios.

    PubMed

    Blatt, Andreas; Bauch, Matthias E; Pörschke, Yvonne; Lohr, Martin

    2015-05-01

    Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and β-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-β-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

  6. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

  7. Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide.

    PubMed

    Severina, I S

    1998-07-01

    In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed. PMID:9721331

  8. Overexpression and characterization of lycopene cyclase (CrtY) from marine bacterium Paracoccus haeundaensis.

    PubMed

    Jeong, Tae Hyug; Ji, Keunho; Kim, Young Tae

    2013-02-01

    Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

  9. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

    PubMed Central

    Gagne, Steve J.; Stout, Jake M.; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M.; Page, Jonathan E.

    2012-01-01

    Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity. PMID:22802619

  10. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

    PubMed

    Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

    2012-07-31

    Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

  11. Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

    PubMed

    Striem, B J; Pace, U; Zehavi, U; Naim, M; Lancet, D

    1989-05-15

    Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

  12. Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm.

    PubMed

    Martin, Emil; Golunski, Eva; Laing, Susan T; Estrera, Anthony L; Sharina, Iraida G

    2014-12-01

    Nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is a key regulator of several important vascular functions and is important for maintaining cardiovascular homeostasis and vascular plasticity. Diminished sGC expression and function contributes to pathogenesis of several cardiovascular diseases. However, the processes that control sGC expression in vascular tissue remain poorly understood. Previous work in animal and cell models revealed the complexity of alternative splicing of sGC genes and demonstrated its importance in modulation of sGC function. The aim of this study was to examine the role of alternative splicing of α1 and β1 sGC in healthy and diseased human vascular tissue. Our study found a variety of α1 and β1 sGC splice forms expressed in human aorta. Their composition and abundance were different between samples of aortic tissue removed during surgical repair of aortic aneurysm and samples of aortas without aneurysm. Aortas with aneurysm demonstrated decreased sGC activity, which correlated with increased expression of dysfunctional sGC splice variants. In addition, the expression of 55-kDa oxidation-resistant α1 isoform B sGC (α1-IsoB) was significantly lower in aortic samples with aneurysm. The α1-IsoB splice variant was demonstrated to support sGC activity in aortic lysates. Together, our results suggest that alternative splicing contributes to diminished sGC function in vascular dysfunction. Precise understanding of sGC splicing regulation could help to design new therapeutic interventions and to personalize sGC-targeting therapies in treatments of vascular disease.

  13. Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases.

    PubMed

    Tremblay, Johanne; Desjardins, Richard; Hum, David; Gutkowska, Jolanta; Hamet, Pavel

    2002-01-01

    Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR- 1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology. PMID:11952095

  14. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  15. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  16. Molecular characterization of tick salivary gland glutaminyl cyclase.

    PubMed

    Adamson, Steven W; Browning, Rebecca E; Chao, Chien-Chung; Bateman, Robert C; Ching, Wei-Mei; Karim, Shahid

    2013-09-01

    Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496

  17. Role of Adenylate Cyclase 1 in Retinofugal Map Development

    PubMed Central

    Dhande, Onkar S.; Bhatt, Shivani; Anishchenko, Anastacia; Elstrott, Justin; Iwasato, Takuji; Swindell, Eric C.; Xu, Hong-Ping; Jamrich, Milan; Itohara, Shigeyoshi; Feller, Marla B.; Crair, Michael C.

    2013-01-01

    The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN. PMID:22102330

  18. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    NASA Technical Reports Server (NTRS)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  19. Saccharinato complexes of Ce(V) with 2-hydroxypyridine: Synthesis, spectroscopic and thermal characteristics of [Ce(sac) 2(SO 4)(H 2O) 4] and [Ce(sac) 2(SO 4)(PyOH) 2

    NASA Astrophysics Data System (ADS)

    Gaballa, Akmal S.; Teleb, Said M.; Müller, Thomas

    2008-10-01

    The synthesis of Ce(IV) complexes [Ce(sac) 2(SO 4)(H 2O) 4] ( 1) and [Ce(sac) 2 (SO 4)(PyOH) 2] ( 2) (sac = saccharinate, PyOH = 2-hydroxypyridine) starting with sodium saccharinate is described. Their vibrational and nuclear magnetic resonance ( 1H, 13C) spectra as well as their thermal mode of degradation were investigated. The data indicate that sac in complex 1 behaves as a monodentate ligand through the nitrogen atoms. Saccharinato ligand in complex 2 shows different mode of coordination, where it behaves as tridentate and binds Ce(IV) through its carbonylic oxygen, nitrogen and sulphonylic oxygen atoms. The most probable structure in complex 2 is that, units of [Ce(sac) 2(SO 4)(PyOH) 2] are linked by bridges of the O- of sac sulphonyl leading to polymeric chains.

  20. Saccharinato complexes of Ce(V) with 2-hydroxypyridine: synthesis, spectroscopic and thermal characteristics of [Ce(sac)2(SO4)(H2O)4] and [Ce(sac)2(SO4)(PyOH)2].

    PubMed

    Gaballa, Akmal S; Teleb, Said M; Müller, Thomas

    2008-10-01

    The synthesis of Ce(IV) complexes [Ce(sac)2(SO4)(H2O)4] (1) and [Ce(sac)2 (SO4)(PyOH)2] (2) (sac=saccharinate, PyOH=2-hydroxypyridine) starting with sodium saccharinate is described. Their vibrational and nuclear magnetic resonance (1H, 13C) spectra as well as their thermal mode of degradation were investigated. The data indicate that sac in complex 1 behaves as a monodentate ligand through the nitrogen atoms. Saccharinato ligand in complex 2 shows different mode of coordination, where it behaves as tridentate and binds Ce(IV) through its carbonylic oxygen, nitrogen and sulphonylic oxygen atoms. The most probable structure in complex 2 is that, units of [Ce(sac)2(SO4)(PyOH)2] are linked by bridges of the O- of sac sulphonyl leading to polymeric chains. PMID:18077205

  1. Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

    PubMed Central

    Purohit, Rahul; Weichsel, Andrzej; Montfort, William R

    2013-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ∼150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction. PMID:23934793

  2. Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase.

    PubMed Central

    Toscano, W A; Westcott, K R; LaPorte, D C; Storm, D R

    1979-01-01

    An adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] preparation that is not stimulated by NaF,5'-guanylyl imidodiphosphate, or Ca2+.calmodulin has been isolated from bovine cerebral cortex by Affi-Gel Blue chromatography and calmodulin-Sepharose chromatography. Sensitivity to these effectors was restored by incubation of the adenylate cyclase preparation with detergent-solubilized protein from bovine cerebral cortex. Reconstitution of of Ca2+.calmodulin activation required the presence of 5'-guanylyl imidodiphosphate. The factor required for restoration of Ca2+.calmodulin stimulation was sensitive to heat, trypsin digestion, and N-ethylmaleimide. These observations suggest that this adenylate cyclase activity requires the presence of one or more guanyl nucleotide binding subunits for calmodulin sensitivity. PMID:293663

  3. Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

    PubMed Central

    Bockaert, J; Deterre, P; Pfister, C; Guillon, G; Chabre, M

    1985-01-01

    Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines. PMID:3861319

  4. In Vivo and Cadaver Studies of the Canalicular/Lacrimal Sac Mucosal Folds

    PubMed Central

    You, Yongsheng; Cao, Jing; Zhang, Xiaogang; Wu, Wencan; Xiao, Tianlin

    2016-01-01

    Purpose. The study aimed to investigate canalicular/lacrimal sac mucosal folds (CLS-MFs) in vivo and in cadavers in order to explore their functional roles in the lacrimal drainage system. Method. The observations of CLS-MFs in vivo were performed on 16 patients with chronic dacryocystitis after undergoing an endonasal endoscopic dacryocystorhinostomy (EE-DCR). The lacrimal sacs and common canaliculi of 19 adult cadavers were dissected. The opening/closing of an orifice and mucosal fold was recorded. All of the specimens were subjected to a histological examination. Results. The upper and lower lacrimal canaliculi in all of the samples united to form a common canaliculus that opened to the lacrimal sac. CLS-MFs were observed in 10 of the 16 patients (62.5%) and 9 of the 19 cadavers (47.4%). The orifices or mucosal folds could be opened or closed when related muscles contracted or relaxed. Histological sections showed a mucosal fold at one side of an orifice. Conclusion. Common canaliculus is the most common type that the canaliculus opens to lacrimal sac. CLS-MFs exist in a certain ratio that can be opened/closed with the movement of the orifices. They may be involved in the drainage of tears or the pathogenesis of acute dacryocystitis or lacrimal sac mucocele. PMID:27242921

  5. Direct Effects of Microalgae and Protists on Herring (Clupea harengus) Yolk Sac Larvae

    PubMed Central

    Illing, Björn; Moyano, Marta; Niemax, Jan; Peck, Myron A.

    2015-01-01

    This study investigated effects of microalgae (Rhodomonas baltica) and heterotrophic protists (Oxyrrhis marina) on the daily growth, activity, condition and feeding success of Atlantic herring (Clupea harengus) larvae from hatch, through the end of the endogenous (yolk sac) period. Yolk sac larvae were reared in the presence and absence of microplankton and, each day, groups of larvae were provided access to copepods. Larvae reared with microalgae and protists exhibited precocious (2 days earlier) and ≥ 60% increased feeding incidence on copepods compared to larvae reared in only seawater (SW). In the absence and presence of microalgae and protists, life span and growth trajectories of yolk sac larvae were similar and digestive enzyme activity (trypsin) and nutritional condition (RNA-DNA ratio) markedly declined in all larvae directly after yolk sac depletion. Thus, microplankton promoted early feeding but was not sufficient to alter life span and growth during the yolk sac phase. Given the importance of early feeding, field programs should place greater emphasis on the protozooplankton-ichthyoplankton link to better understand match-mismatch dynamics and bottom-up drivers of year class success in marine fish. PMID:26035592

  6. Rectal sac distention is induced by 20-hydroxyecdysone in the pupa of Bombyx mori.

    PubMed

    Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

    2009-03-01

    Holometabolous insects do not excrete but store metabolic wastes during the pupal period. The waste is called meconium and is purged after adult emergence. Although the contents of meconium are well-studied, the developmental and physiological regulation of meconium accumulation is poorly understood. In Bombyx mori, meconium is accumulated in the rectal sac; thereby, the rectal sac distends at the late pupal stage. Here, we show that rectal sac distention occurs between 4 and 5 days after pupation. The distention is halted by brain-removal just after larval-pupal ecdysis but not by brain-removal 1 day after pupation. In the pupae, brain-removal just after ecdysis kept the hemolymph ecdysteroid titer low during early and mid-pupal stages. An injection of 20-hydroxyecdysone (20E) evoked the distention that was halted by brain-removal in a dose-dependent manner. Therefore, brain-removal caused the lack of ecdysteroid, and rectal sac distention did not appear in the brain-removed pupae because of the lack of ecdysteroid. We conclude that rectal sac distention is one of the developmental events regulated by 20E during the pupal period in B. mori.

  7. Sac-0601 prevents retinal vascular leakage in a mouse model of diabetic retinopathy.

    PubMed

    Maharjan, Sony; Lee, Sujin; Agrawal, Vijayendra; Choi, Hyun-Jung; Maeng, Yong-Sun; Kim, Kyeojin; Kim, Nam-Jung; Suh, Young-Ger; Kwon, Young-Guen

    2011-04-25

    Endothelium integrity is important for the normal functioning of vessels, the disruption of which can lead to disease. The blood-retinal barrier required for normal retinal function is compromised in diabetic retinopathy, causing retinal vascular leakage. Previously, we demonstrated the ability of Sac-0601[((2R,3S)-3-acetoxy-6-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-2H-pyran-2-yl)methyl acetate], a pseudo-sugar derivative of cholesterol, to increase survival of retinal endothelial cells. In the present study, we evaluated the ability of Sac-0601 to prevent retinal vascular leakages in vitro and in vivo. Sac-0601 treatment blocked VEGF-induced formation of actin stress fibers and stabilized the cortical actin ring in retinal endothelial cells. It also inhibited degradation of occludin, an important tight junction protein, and blocked VEGF-induced disruption of its linear pattern at the cell border. The [(14)C] sucrose permeability assay demonstrated that Sac-0601 was able to prevent VEGF-induced retinal endothelial permeability. The compound inhibited the vascular leakage in retina of mice intravitreally injected with VEGF. And it also significantly reduced the leakage in retina of diabetic retinopathy mice model. Taken together, our findings suggest the potential therapeutic usefulness of Sac-0601 for retinal vascular permeability diseases.

  8. Direct Effects of Microalgae and Protists on Herring (Clupea harengus) Yolk Sac Larvae.

    PubMed

    Illing, Björn; Moyano, Marta; Niemax, Jan; Peck, Myron A

    2015-01-01

    This study investigated effects of microalgae (Rhodomonas baltica) and heterotrophic protists (Oxyrrhis marina) on the daily growth, activity, condition and feeding success of Atlantic herring (Clupea harengus) larvae from hatch, through the end of the endogenous (yolk sac) period. Yolk sac larvae were reared in the presence and absence of microplankton and, each day, groups of larvae were provided access to copepods. Larvae reared with microalgae and protists exhibited precocious (2 days earlier) and ≥ 60% increased feeding incidence on copepods compared to larvae reared in only seawater (SW). In the absence and presence of microalgae and protists, life span and growth trajectories of yolk sac larvae were similar and digestive enzyme activity (trypsin) and nutritional condition (RNA-DNA ratio) markedly declined in all larvae directly after yolk sac depletion. Thus, microplankton promoted early feeding but was not sufficient to alter life span and growth during the yolk sac phase. Given the importance of early feeding, field programs should place greater emphasis on the protozooplankton-ichthyoplankton link to better understand match-mismatch dynamics and bottom-up drivers of year class success in marine fish.

  9. "Gestation sac" volumes as determined by sonar in the first trimester of pregnancy.

    PubMed

    Robinson, H P

    1975-02-01

    Sonar estimates of "gestation sac" volumes in the first trimester of pregnancy were made from a series of 319 measurements; the volumes estimated included the amniotic fluid, the extraembryonic coelom and the fetus. The technique employed involved the use of parallel section scans taken in series from one end of the gestation sac to the other, followed by planimetric measurements of the sac areas so produced. The potential errors which may be incurred in these measurements are discussed, and it is considered that the technique carries an overall possible error in the order of plus or minus 10 per cent. Growth curves produced from the measurements in this series show that the sac increases in volume from a mean of 1 ml. at 6 weeks to a mean of 100 ml. at 13 weeks, initially in an exponential fashion but latterly in a more linear manner. The mean values of the sonar gestation sac fluid volumes (after subtraction of the estimated fetal volume) in the 10 to 13 week range, show good correlation with those amniotic fluid values reported in the literature where direct measurements were made at the time of hysterotomy. As a method of assessing the maturity of a pregnancy this technique is of lesser value than the sonar measurement of fetal crown-rump length because of the relatively wider scatter of results. It has, however, found a useful place in clinical practice in the early sonar diagnosis of blighted ova or anembryonic pregnancies.

  10. A status report on the development of SAC2000: A new seismic analysis code

    SciTech Connect

    Goldstein, P.; Minner, L.

    1995-08-01

    We are developing a new Seismic Analysis Code (SAC2000) that will meet the research needs of the seismic research and treaty monitoring communities. Our first step in this development was to rewrite the original Seismic Analysis Code (SAC) -- a Fortran code that was approximately 140,000 lines long -- in the C programming language. This rewrite has resulted in a much more robust code that is faster, more efficient, and more portable than the original. We have implemented important processing capabilities such as convolution and binary monograms, and we have significantly enhanced several previously existing capabilities. For example, the spectrogram command now produces a correctly registered plot of the input time series and a color image of the output spectrogram. We have also added an image plotting capability with access to 17 predefined color tables or custom color tables. A rewritten version of the readcss command can now be used to access any of the documented css.3.0 database data formats, a capability that is particularly important to the Air Force Technical Applications Center (AFTAC) and the monitoring community. A much less visible, but extremely important contribution is the correction of numerous inconsistencies and errors that have evolved because of piecemeal development and limited maintenance since SAC was first written. We have also incorporated on-line documentation and have made SAC documentation available on the Internet via the world-wide-web at http://www-ep/tvp/sac.html.

  11. Direct Effects of Microalgae and Protists on Herring (Clupea harengus) Yolk Sac Larvae.

    PubMed

    Illing, Björn; Moyano, Marta; Niemax, Jan; Peck, Myron A

    2015-01-01

    This study investigated effects of microalgae (Rhodomonas baltica) and heterotrophic protists (Oxyrrhis marina) on the daily growth, activity, condition and feeding success of Atlantic herring (Clupea harengus) larvae from hatch, through the end of the endogenous (yolk sac) period. Yolk sac larvae were reared in the presence and absence of microplankton and, each day, groups of larvae were provided access to copepods. Larvae reared with microalgae and protists exhibited precocious (2 days earlier) and ≥ 60% increased feeding incidence on copepods compared to larvae reared in only seawater (SW). In the absence and presence of microalgae and protists, life span and growth trajectories of yolk sac larvae were similar and digestive enzyme activity (trypsin) and nutritional condition (RNA-DNA ratio) markedly declined in all larvae directly after yolk sac depletion. Thus, microplankton promoted early feeding but was not sufficient to alter life span and growth during the yolk sac phase. Given the importance of early feeding, field programs should place greater emphasis on the protozooplankton-ichthyoplankton link to better understand match-mismatch dynamics and bottom-up drivers of year class success in marine fish. PMID:26035592

  12. Developmental expression of otoconin-22 in the bullfrog endolymphatic sac and inner ear.

    PubMed

    Yaoi, Yuichi; Onda, Tomoaki; Hidaka, Yoshie; Yajima, Shinya; Suzuki, Masakazu; Tanaka, Shigeyasu

    2004-05-01

    In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear. PMID:15100243

  13. Changed sensitivity of adenylate cyclase signaling system to biogenic amines and peptide hormones in tissues of starving rats.

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-07-01

    In the myocardium and skeletal muscles of rats deprived of food for 2 days, basal activity of adenylate cyclase decreased, while the sensitivity of adenylate cyclase signaling system to the stimulating effects of non-hormonal agents (guanine nucleotides and NaF) and beta-agonist isoproterinol modulating adenylate cyclase through stimulating G proteins increased. In starving organism, the regulatory effects of hormones realizing their effects through inhibitory G proteins (somatostatin in the myocardium and bromocryptin in the brain) weakened. Their inhibitory effects on forskolin-stimulated adenylate cyclase activity and stimulating effects on binding of guanosine triphosphate decreased. In the brain of starving rats, the differences in the sensitivity of the adenylate cyclase signaling system to hormones and nonhormonal agents were less pronounced than in the muscle tissues, which attested to tissue-specific changes in the functional state of this system under conditions of 2-day starvation.

  14. User's guide to SAC, a computer program for computing discharge by slope-area method

    USGS Publications Warehouse

    Fulford, Janice M.

    1994-01-01

    This user's guide contains information on using the slope-area program, SAC. SAC can be used to compute peak flood discharges from measurements of high-water marks along a stream reach. The Slope-area method used by the program is the U.S. Geological Survey (USGS) procedure presented in Techniques of Water Resources Investigations of the U.S. Geological Survey, beok 3, chapter A2, "Measurement of Peak Discharge by the Slope-Area Method." The program uses input files that have formats compatible with those used by the water-surface profile program (WSPRO) described in the Federal Highways Administration publication FHWA-IP-89-027. The guide briefly describes the slope-area method documents the input requirements and the output produced, and demonstrates use of SAC.

  15. Anisotropic shrinkage of insect air sacs revealed in vivo by X-ray microtomography.

    PubMed

    Xu, Liang; Chen, Rongchang; Du, Guohao; Yang, Yiming; Wang, Feixiang; Deng, Biao; Xie, Honglan; Xiao, Tiqiao

    2016-01-01

    Air sacs are thought to be the bellows for insect respiration. However, their exact mechanism of action as a bellows remains unclear. A direct way to investigate this problem is in vivo observation of the changes in their three-dimensional structures. Therefore, four-dimensional X-ray phase contrast microtomography is employed to solve this puzzle. Quantitative analysis of three-dimensional image series reveals that the compression of the air sac during respiration in bell crickets exhibits obvious anisotropic characteristics both longitudinally and transversely. Volumetric changes of the tracheal trunks in the prothorax further strengthen the evidence of this finding. As a result, we conclude that the shrinkage and expansion of the insect air sac is anisotropic, contrary to the hypothesis of isotropy, thereby providing new knowledge for further research on the insect respiratory system. PMID:27580585

  16. [Chemotherapy of yolk sac tumor heterotransplanted to nude mice (author's transl)].

    PubMed

    Sawada, M; Hayakawa, K; Matsui, Y; Nishiura, H; Okudaira, Y

    1980-10-01

    Chemotherapy of yolk sac tumor heterotransplanted to nude mice was studied. 1. Yolk sac tumor of the ovary taken from a 38-year -old woman was transplanted to BALB/c female nude mice. The transplantable tumor cells produce a solid tumor, designated as YST-1 tumor. The YST-1 tumor cells preserve the histological appearance of a human yolk sac tumor and produce x-fetoprotein. The tumors on passage 8 were used for experimental chemotherapy. 2. Anticancer drugs clinically known to be effective for ovarian cancer, such as Adriamycin, Carbazilquinone, 5-Fluorouracil, Cyclophosphamide, Mitomycin C, Chromomycin A3, Vinblastine and Bleomycin were administered intraperitoneally to tumor-bearing nude mice. Tumor size was measured two or three times a week during the course of experiments. Therapeutic effects were evaluated by tumor size and relative tumor size before and after experiments. Among these drugs, Vinblastine and Bleomycin combination showed the significant effect arresting the growth of YST-1 tumor.

  17. Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

    PubMed

    Isogai, Masamichi; Yoshida, Tetu; Shimura, Takuya; Yoshikawa, Nobuyuki

    2015-10-01

    We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

  18. A New Technique for Reduction the Phase Induced Intensity Noise in SAC-OCDMA Systems

    NASA Astrophysics Data System (ADS)

    Abd, Thanaa Hussein; Aljunid, Syed Alwee; Fadhil, Hilal Adnan

    2011-12-01

    A new code for reduction the phase induced intensity noise has been presented. The new code is proposed for Spectral Amplitude-Coding Optical Code Division Multiple Accesses (SAC-OCDMA). This new code family we call it Dynamic Cyclic Shift (DCS) code. The DCS code reduced the effect of Multi Access Interference (MAI) due to it is the property of variable cross correlation. We find that the performance of the DCS code is a batter than other SAC-OCDMA codes such as; Random Diagonal (RD) code, Modified Quadratic Congruence (MQC) code and Modified Frequency Hopping (MFH) code. Through the mathematical calculation and simulation analysis, for the bit-error rate of DCS code is significantly better than other SAC-OCDMA codes, the effect of Phase Induced Intensity Noise is reduced. In addition, proofof-principle simulations of 10 Gb/s for 20 km have been successfully demonstrated and achieved low BER compared to the other codes.

  19. Anisotropic shrinkage of insect air sacs revealed in vivo by X-ray microtomography

    NASA Astrophysics Data System (ADS)

    Xu, Liang; Chen, Rongchang; Du, Guohao; Yang, Yiming; Wang, Feixiang; Deng, Biao; Xie, Honglan; Xiao, Tiqiao

    2016-09-01

    Air sacs are thought to be the bellows for insect respiration. However, their exact mechanism of action as a bellows remains unclear. A direct way to investigate this problem is in vivo observation of the changes in their three-dimensional structures. Therefore, four-dimensional X-ray phase contrast microtomography is employed to solve this puzzle. Quantitative analysis of three-dimensional image series reveals that the compression of the air sac during respiration in bell crickets exhibits obvious anisotropic characteristics both longitudinally and transversely. Volumetric changes of the tracheal trunks in the prothorax further strengthen the evidence of this finding. As a result, we conclude that the shrinkage and expansion of the insect air sac is anisotropic, contrary to the hypothesis of isotropy, thereby providing new knowledge for further research on the insect respiratory system.

  20. Amniote yolk sacs: diversity in reptiles and a hypothesis on their origin.

    PubMed

    Elinson, Richard P; Stewart, James R; Bonneau, Laurie J; Blackburn, Daniel G

    2014-01-01

    Oviparous amniotes produce a large yolky egg that gives rise to a free-living hatchling. Structural characteristics and functional attributes of the egg are best known for birds, which have a large mass of fluid yolk surrounded by an extraembryonic yolk sac. Yolk nutrients are delivered to the embryo via the vascular yolk sac. This developmental pattern and nutrient transport mechanism is thought to be representative of all other lineages of amniotes. Recent discovery of a snake with cellularized yolk organized around a meshwork of blood vessels reveals an additional pattern for yolk mobilization, which may also occur in other squamate reptiles (lizards and snakes). This complex yolk sac raises interesting questions about developmental mechanisms and suggests a possible model for the transition between the egg of anamniotes and that of amniotes.

  1. Anisotropic shrinkage of insect air sacs revealed in vivo by X-ray microtomography

    PubMed Central

    Xu, Liang; Chen, Rongchang; Du, Guohao; Yang, Yiming; Wang, Feixiang; Deng, Biao; Xie, Honglan; Xiao, Tiqiao

    2016-01-01

    Air sacs are thought to be the bellows for insect respiration. However, their exact mechanism of action as a bellows remains unclear. A direct way to investigate this problem is in vivo observation of the changes in their three-dimensional structures. Therefore, four-dimensional X-ray phase contrast microtomography is employed to solve this puzzle. Quantitative analysis of three-dimensional image series reveals that the compression of the air sac during respiration in bell crickets exhibits obvious anisotropic characteristics both longitudinally and transversely. Volumetric changes of the tracheal trunks in the prothorax further strengthen the evidence of this finding. As a result, we conclude that the shrinkage and expansion of the insect air sac is anisotropic, contrary to the hypothesis of isotropy, thereby providing new knowledge for further research on the insect respiratory system. PMID:27580585

  2. Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

    PubMed

    Isogai, Masamichi; Yoshida, Tetu; Shimura, Takuya; Yoshikawa, Nobuyuki

    2015-10-01

    We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs. PMID:26176979

  3. Postcranial skeletal pneumaticity and air-sacs in the earliest pterosaurs

    PubMed Central

    Butler, Richard J.; Barrett, Paul M.; Gower, David J.

    2009-01-01

    Patterns of postcranial skeletal pneumatization (PSP) indicate that pterosaurs possessed components of a bird-like respiratory system, including a series of ventilatory air-sacs. However, the presence of PSP in the oldest known pterosaurs has not been unambiguously demonstrated by previous studies. Here we provide the first unequivocal documentation of PSP in Late Triassic and earliest Jurassic pterosaurs. This demonstrates that PSP and, by inference, air-sacs were probably present in the common ancestor of almost all known pterosaurs, and has broader implications for the evolution of respiratory systems in bird-line archosaurs, including dinosaurs. PMID:19411265

  4. Endolymphatic Sac Tumor Showing Increased Activity on 68Ga DOTATATE PET/CT.

    PubMed

    Papadakis, Georgios Z; Millo, Corina; Sadowski, Samira M; Bagci, Ulas; Patronas, Nicholas J

    2016-10-01

    Endolymphatic sac tumors (ELSTs) are rare tumors arising from the epithelium of the endolymphatic sac and duct that can be either sporadic or associated with von Hippel-Lindau (VHL) disease. We report a case of a VHL patient with histologically proven residual ELST who underwent Ga DOTATATE PET/CT showing increased activity (SUVmax, 6.29) by the ELST. The presented case of a VHL-associated ELST with increased Ga DOTATATE uptake indicates cell-surface expression of somatostatin receptors by this tumor, suggesting the potential application of somatostatin receptor imaging using Ga DOTA-conjugated peptides in the workup and management of these patients. PMID:27454593

  5. Soluble guanylyl cyclase is involved in PDT-induced injury of crayfish glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, V. D.; Uzdensky, A. B.

    2016-04-01

    Photodynamic therapy (PDT) is a potential tool for selective destruction of malignant brain tumors. However, not only malignant but also healthy neurons and glial cells may be damaged during PDT. Nitric oxide is an important modulator of cell viability and intercellular neuroglial communications. NO have been already shown to participate in PDT-induced injury of neurons and glial cells. As soluble guanylyl cyclase is the only known receptor for NO, we have studied the possible role of soluble guanylyl cyclase in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Using inhibitory analysis we have shown that during PDT soluble guanylyl cyclase, probably, has proapoptotic and antinecrotic effect on the glial cells of the isolated crayfish stretch receptor. Proapoptotic effect of soluble guanylyl cyclase could be mediated by protein kinase G (PKG). Thus, the involvement of NO/sGC/cGMP/PKG signaling pathway in PDT-induced apoptosis of glial cells was indirectly demonstrated.

  6. Defective responsiveness of adenylate cyclase to forskolin in the Drosophila memory mutant rutabaga.

    PubMed

    Dudai, Y; Sher, B; Segal, D; Yovell, Y

    1985-12-01

    The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopulation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the Km of the enzyme for free Mg2+ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca2+-stimulation of adenylate cyclase in rut17. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed. PMID:3935769

  7. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  8. Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes.

    PubMed

    Tolkovsky, A M; Levitzki, A

    1978-09-01

    The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a

  9. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure.

    PubMed

    Scarpace, P J; Baresi, L A; Morley, J E

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the beta-adrenergic pathway, adenylate cyclase activity and beta-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. beta-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed beta 1- and beta 2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in beta-adrenergic receptor density due to a loss of the beta 1-adrenergic subtype. This BAT beta-adrenergic receptor downregulation was tissue specific, since myocardial beta-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. In contrast, food deprivation did not alter BAT beta-adrenergic receptor density. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. The ratio of isoproterenol-stimulated to forskolin-stimulated adenylate cyclase activity decreased in the sucrose-fed and cold-exposed rats but not in the food-deprived rats. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of beta-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability. PMID:2827501

  10. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

    PubMed Central

    Cunningham, F X; Sun, Z; Chamovitz, D; Hirschberg, J; Gantt, E

    1994-01-01

    A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme. PMID:7919981

  11. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  12. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  13. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    PubMed

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  14. Lethality of glnD null mutations in Azotobacter vinelandii is suppressible by prevention of glutamine synthetase adenylylation.

    PubMed

    Colnaghi, R; Rudnick, P; He, L; Green, A; Yan, D; Larson, E; Kennedy, C

    2001-05-01

    GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridylylates two related proteins, PII and GlnK. The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC. Results presented here demonstrate that glnD is an essential gene in Azotobacter vinelandii. Null glnD mutations were introduced into the A. vinelandii genome, but none could be stably maintained unless a second mutation was present that resulted in unregulated activity of GS. One mutation, gln-71, occurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein. The second, created by design, was glnAY407F (MV75), altering the adenylylation site of GS. The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia coli glnE gene into MV72, a glnD(+) derivative of MV71, restored the regulation of GS activity. GlnK-UMP is therefore apparently required for GS to be sufficiently deadenylylated in A. vinelandii for growth to occur. The DeltaglnD GS(c) isolates were Nif(-), which could be corrected by introducing a nifL mutation, confirming a role for GlnD in mediating nif gene regulation via some aspect of the NifL/NifA interaction. MV71 was unexpectedly NtrC(+), suggesting that A. vinelandii NtrC activity might be regulated differently than in enteric organisms.

  15. Modeling the Rate-Dependent Durability of Reduced-Ag SAC Interconnects for Area Array Packages Under Torsion Loads

    NASA Astrophysics Data System (ADS)

    Srinivas, Vikram; Menon, Sandeep; Osterman, Michael; Pecht, Michael G.

    2013-08-01

    Solder durability models frequently focus on the applied strain range; however, the rate of applied loading, or strain rate, is also important. In this study, an approach to incorporate strain rate dependency into durability estimation for solder interconnects is examined. Failure data were collected for SAC105 solder ball grid arrays assembled with SAC305 solder that were subjected to displacement-controlled torsion loads. Strain-rate-dependent (Johnson-Cook model) and strain-rate-independent elastic-plastic properties were used to model the solders in finite-element simulation. Test data were then used to extract damage model constants for the reduced-Ag SAC solder. A generalized Coffin-Manson damage model was used to estimate the durability. The mechanical fatigue durability curve for reduced-silver SAC solder was generated and compared with durability curves for SAC305 and Sn-Pb from the literature.

  16. Dispensability of the SAC Depends on the Time Window Required by Aurora B to Ensure Chromosome Biorientation

    PubMed Central

    Monje-Casas, Fernando

    2015-01-01

    Aurora B and the spindle assembly checkpoint (SAC) collaborate to ensure the proper biorientation of chromosomes during mitosis. However, lack of Aurora B activity and inactivation of the SAC have a very different impact on chromosome segregation. This is most evident in Saccharomyces cerevisiae, since in this organism the lack of Aurora B is lethal and leads to severe aneuploidy problems, while the SAC is dispensable under normal growth conditions and mutants in this checkpoint do not show evident chromosome segregation defects. We demonstrate that the efficient repair of incorrect chromosome attachments by Aurora B during the initial stages of spindle assembly in budding yeast determines the lack of chromosome segregation defects in SAC mutants, and propose that the differential time window that Aurora B kinase requires to establish chromosome biorientation is the key factor that determines why some cells are more dependent on a functional SAC than others. PMID:26661752

  17. The corn snake yolk sac becomes a solid tissue filled with blood vessels and yolk-rich endodermal cells

    PubMed Central

    Elinson, Richard P.; Stewart, James R.

    2014-01-01

    The amniote egg was a key innovation in vertebrate evolution because it supports an independent existence in terrestrial environments. The egg is provisioned with yolk, and development depends on the yolk sac for the mobilization of nutrients. We have examined the yolk sac of the corn snake Pantherophis guttatus by the dissection of living eggs. In contrast to the familiar fluid-filled sac of birds, the corn snake yolk sac invades the yolk mass to become a solid tissue. There is extensive proliferation of yolk-filled endodermal cells, which associate with a meshwork of blood vessels. These novel attributes of the yolk sac of corn snakes compared with birds suggest new pathways for the evolution of the amniote egg. PMID:24402715

  18. Effects of granulation on organic acid metabolism and its relation to mineral elements in Citrus grandis juice sacs.

    PubMed

    Wang, Xian-You; Wang, Ping; Qi, Yi-Ping; Zhou, Chen-Ping; Yang, Lin-Tong; Liao, Xin-Yan; Wang, Liu-Qing; Zhu, Dong-Huang; Chen, Li-Song

    2014-02-15

    We investigated the effects of granulation on organic acid metabolism and its relation to mineral elements in 'Guanximiyou' pummelo (Citrus grandis) juice sacs. Granulated juice sacs had decreased concentrations of citrate and isocitrate, thus lowering juice sac acidity. By contrast, malate concentration was higher in granulated juice sacs than in normal ones. The reduction in citrate concentration might be caused by increased degradation, as indicated by enhanced aconitase activity, whilst the increase in malate concentration might be caused by increased biosynthesis, as indicated by enhanced phosphoenolpyruvate carboxylase (PEPC). Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that the activities of most acid-metabolizing enzymes were regulated at the transcriptional level, whilst post-translational modifications might influence the PEPC activity. Granulation led to increased accumulation of mineral elements (especially phosphorus, magnesium, sulphur, zinc and copper) in juice sacs, which might be involved in the incidence of granulation in pummelo fruits.

  19. 2 CFR Appendix X to Part 200 - Data Collection Form (Form SF-SAC)

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Data Collection Form (Form SF-SAC) X Appendix X to Part 200 Grants and Agreements Office of Management and Budget Guidance for Grants and... PRINCIPLES, AND AUDIT REQUIREMENTS FOR FEDERAL AWARDS Pt. 200, App. X Appendix X to Part 200—Data...

  20. Yolk sac tumor of the seminal vesicles: A rare malignant cause of hematospermia.

    PubMed

    Gill, Jonathan D; Bhattarai, Selina; Patel, Chirag N; Paul, Alan B

    2015-01-01

    Extra-gonadal yolk sac tumors (YSTs) are rare and generally associated with poor outcomes. Involvement of the seminal vesicles is extremely rare with only one previously described case. We report a case of a primary YST of the seminal vesicles and discuss the management strategy.

  1. Ventilation patterns of the songbird lung/air sac system during different behaviors

    PubMed Central

    Mackelprang, Rebecca; Goller, Franz

    2013-01-01

    SUMMARY Unidirectional, continuous airflow through the avian lung is achieved through an elaborate air sac system with a sequential, posterior to anterior ventilation pattern. This classical model was established through various approaches spanning passively ventilated systems to mass spectrometry analysis of tracer gas flow into various air sacs during spontaneous breathing in restrained ducks. Information on flow patterns in other bird taxa is missing, and these techniques do not permit direct tests of whether the basic flow pattern can change during different behaviors. Here we use thermistors implanted into various locations of the respiratory system to detect small pulses of tracer gas (helium) to reconstruct airflow patterns in quietly breathing and behaving (calling, wing flapping) songbirds (zebra finch and yellow-headed blackbird). The results illustrate that the basic pattern of airflow in these two species is largely consistent with the model. However, two notable differences emerged. First, some tracer gas arrived in the anterior set of air sacs during the inspiration during which it was inhaled, suggesting a more rapid throughput through the lung than previously assumed. Second, differences in ventilation between the two anterior air sacs emerged during calling and wing flapping, indicating that adjustments in the flow pattern occur during dynamic behaviors. It is unclear whether this modulation in ventilation pattern is passive or active. This technique for studying ventilation patterns during dynamic behaviors proves useful for establishing detailed timing of airflow and modulation of ventilation in the avian respiratory system. PMID:23788706

  2. Ventilation patterns of the songbird lung/air sac system during different behaviors.

    PubMed

    Mackelprang, Rebecca; Goller, Franz

    2013-10-01

    Unidirectional, continuous airflow through the avian lung is achieved through an elaborate air sac system with a sequential, posterior to anterior ventilation pattern. This classical model was established through various approaches spanning passively ventilated systems to mass spectrometry analysis of tracer gas flow into various air sacs during spontaneous breathing in restrained ducks. Information on flow patterns in other bird taxa is missing, and these techniques do not permit direct tests of whether the basic flow pattern can change during different behaviors. Here we use thermistors implanted into various locations of the respiratory system to detect small pulses of tracer gas (helium) to reconstruct airflow patterns in quietly breathing and behaving (calling, wing flapping) songbirds (zebra finch and yellow-headed blackbird). The results illustrate that the basic pattern of airflow in these two species is largely consistent with the model. However, two notable differences emerged. First, some tracer gas arrived in the anterior set of air sacs during the inspiration during which it was inhaled, suggesting a more rapid throughput through the lung than previously assumed. Second, differences in ventilation between the two anterior air sacs emerged during calling and wing flapping, indicating that adjustments in the flow pattern occur during dynamic behaviors. It is unclear whether this modulation in ventilation pattern is passive or active. This technique for studying ventilation patterns during dynamic behaviors proves useful for establishing detailed timing of airflow and modulation of ventilation in the avian respiratory system.

  3. Testicular yolk sac tumor and impaired spermatogenesis in a Holstein Friesian calf.

    PubMed

    Schindewolffs, Lia; Dierks, Claudia; Heppelmann, Maike; Gähle, Marion; Piechotta, Marion; Beineke, Andreas; Brehm, Ralph; Distl, Ottmar

    2015-01-01

    Yolk sac tumors are testicular germ-cell tumors of the non-seminoma type. In cattle, this neoplasm is very rare and to date has only been described three times. In human males, it usually occurs in infants and children. Immunohistochemistry provides solid criteria for diagnostics. Especially present pathognomonic Schiller-Duval bodies are helpful for identification. In this report, a 32-day-old Holstein Friesian calf presented with a highly enlarged right testis. Sonographic examination was performed and blood samples were taken to measure testosterone and estrogen levels. Furthermore, the testis was surgically removed and macroscopically, histologically, and immunohistochemically examined which lead to the diagnosis of testicular yolk sac tumor. The second testis was descended until the age of nine months and histology revealed impaired spermatogenesis. This report provides the first sonographic images of bovine testicular yolk sac tumor as well as the first information about hormone levels in calves with this neoplasm. It also shows the importance to combine anamnesis, histomorphological, and immunohistochemical findings in order to diagnose yolk sac tumors when pathognomonic structures are not present.

  4. 48 CFR 301.603-72 - FAC-C and HHS SAC certification requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false FAC-C and HHS SAC certification requirements. 301.603-72 Section 301.603-72 Federal Acquisition Regulations System HEALTH AND HUMAN SERVICES GENERAL HHS ACQUISITION REGULATION SYSTEM Career Development, Contracting Authority,...

  5. 38 CFR Appendix A to Part 41 - Data Collection Form (Form SF-SAC)

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Data Collection Form (Form SF-SAC) A Appendix A to Part 41 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) AUDITS OF STATES, LOCAL GOVERNMENTS, AND NON-PROFIT ORGANIZATIONS Pt. 41, App. A,...

  6. Bleomycin-Induced Flagellate Erythema in a Patient Diagnosed with Ovarian Yolk Sac Tumor

    PubMed Central

    Boussios, Stergios; Moschetta, Michele; McLachlan, Jennifer; Banerjee, Susana

    2015-01-01

    Flagellate linear hyperpigmentation can rarely be caused by the chemotherapy agent, bleomycin. Herein, we describe the case of a 20-year-old woman treated with bleomycin for an ovarian yolk sac tumor and review the prominent features of this form of dermatitis. PMID:26798532

  7. Cells of the connective tissue differentiate and migrate into pollen sacs

    NASA Astrophysics Data System (ADS)

    Iqbal, M. C. M.; Wijesekara, Kolitha B.

    2002-01-01

    In angiosperms, archesporial cells in the anther primordium undergo meiosis to form haploid pollen, the sole occupants of anther sacs. Anther sacs are held together by a matrix of parenchyma cells, the connective tissue. Cells of the connective tissue are not known to differentiate. We report the differentiation of parenchyma cells in the connective tissue of two Gordonia species into pollen-like structures (described as pseudopollen), which migrate into the anther sacs before dehiscence. Pollen and pseudopollen were distinguishable by morphology and staining. Pollen were tricolpate to spherical while pseudopollen were less rigid and transparent with a ribbed surface. Both types were different in size, shape, staining and surface architecture. The ratio of the number of pseudopollen to pollen was 1:3. During ontogeny in the connective tissue, neither cell division nor tetrad formation was observed and hence pseudopollen were presumed to be diploid. Only normal pollen germinated on a germination medium. Fixed preparations in time seemed to indicate that pseudopollen migrate from the connective tissue into the anther sac.

  8. An observing system simulation experiment (OSSE) for the aquarius/SAC-D soil moisture product

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An Observing System Simulation Experiment for the Aquarius/SAC-D mission has been developed for assessing the accuracy of soil moisture retrievals from passive L-band remote sensing. The implementation of the OSSE is based on: a 1-km land surface model over the Red-Arkansas River Basin, a forward mi...

  9. Entire lacrimal sac within the ethmoid sinus: outcomes of powered endoscopic dacryocystorhinostomy

    PubMed Central

    Ali, Mohammad Javed; Singh, Swati; Naik, Milind N

    2016-01-01

    Background The aim of this study was to report the outcomes of powered endoscopic dacryocystorhinostomy (PEnDCR) in patients with lacrimal sac within the sinus. Materials and methods Retrospective analysis was performed on all patients who underwent PEnDCR and were intraoperatively documented to have complete lacrimal sac in sinus. Data collected included demographics, clinical presentations, associated lacrimal and nasal anomalies, intraoperative findings, intraoperative guidance, complications, postoperative ostium behavior, and anatomical and functional success. A minimum follow-up of 6 months postsurgery was considered for final analysis. Results A total of 17 eyes of 15 patients underwent PEnDCR using standard protocols, but with additional intraoperative guidance where required and careful maneuvering in the ethmoid sinus. The mean age of the patients was 37.2 (range 17–60) years. Of the unilateral cases, 69% (nine of 13) showed left-side predisposition; 80% of patients showed regurgitation on pressure over the lacrimal sac area. Associated lacrimal and nasal anomalies were observed in 13.3% (two of 15) and 40% (six of 15), respectively. At a mean follow-up of 6.6 months, anatomical and functional success were observed in 93.3% (14 of 15). One patient showed failure secondary to cicatricial closure of the ostium. Conclusion An entire sac within an ethmoid sinus poses a surgical challenge. Good sinus-surgery training, thorough knowledge of endoscopic anatomy, careful maneuvering, and use of intraoperative navigation guidance result in good outcomes with PEnDCR. PMID:27462137

  10. Regulation of ascorbic acid metabolism by blue LED light irradiation in citrus juice sacs.

    PubMed

    Zhang, Lancui; Ma, Gang; Yamawaki, Kazuki; Ikoma, Yoshinori; Matsumoto, Hikaru; Yoshioka, Terutaka; Ohta, Satoshi; Kato, Masaya

    2015-04-01

    In the present study, the effects of red and blue LED lights on the accumulation of ascorbic acid (AsA) were investigated in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon. The results showed that the blue LED light treatment effectively increased the AsA content in the juice sacs of the three citrus varieties, whereas the red LED light treatment did not. By increasing the blue LED light intensity, the juice sacs of the three citrus varieties accumulated more AsA. Moreover, continuous irradiation with blue LED light was more effective than pulsed irradiation for increasing the AsA content in the juice sacs of the three citrus varieties. Gene expression results showed that the modulation of AsA accumulation by blue LED light was highly regulated at the transcription level. The up-regulation of AsA biosynthetic genes (CitVTC1, CitVTC2, CitVTC4, and CitGLDH), AsA regeneration genes (CitMDAR1, CitMDAR2, and CitDHAR) and two GSH-producing genes (CitGR and CitchGR) contributed to these increases in the AsA content in the three citrus varieties.

  11. Late Sac Behavior after Endovascular Aneurysm Repair for Abdominal Aortic Aneurysm

    PubMed Central

    Okada, Masahiro; Onohara, Toshihiro; Okamoto, Minoru; Yamamoto, Tsuyoshi; Shimoe, Yasushi; Yamashita, Masafumi; Takahashi, Toshiki; Kishimoto, Jyunji; Mizuno, Akihiro; Kei, Junichi; Nakai, Mikizou; Sakaki, Masayuki; Suhara, Hitoshi; Kasashima, Fuminori; Endo, Masamitsu; Nishina, Takeshi; Furuyama, Tadashi; Kawasaki, Masakazu; Iwata, Keiji; Marumoto, Akira; Urata, Yasuhisa; Sato, Katsutoshi; Ryugo, Masahiro

    2016-01-01

    Background: Sac behavior after endovascular aneurysm repair (EVAR) for abdominal aortic aneurysms (AAAs) is considered as a surrogate for the risk of late rupture. The purpose of the study is to assess the sac behavior of AAAs after EVAR. Methods and Results: Late sac enlargement (LSE) (≥5 mm) and late sac shrinkage (LSS) (≥5 mm) were analyzed in 589 consecutive patients who were registered at 14 national centers in Japan. The proportions of patients who had LSE at 1, 3 and 5 years were 2.6% ± 0.7%, 10.0% ± 1.6% and 19.0% ± 2.9%. The proportions of patients who had LSS at 1, 3 and 5 years were 50.1% ± 0.7%, 59.2% ± 2.3% and 61.7% ± 2.7%. Multiple logistic regression analysis identified two variables as a risk factor for LSE; persistent endoleak (Odds ratio 9.56 (4.84–19.49), P <0.001) and low platelet count (Odds ratio 0.92 (0.86–0.99), P = 0.0224). The leading cause of endoleak in patients with LSE was type II. Conclusions: The incidence of LSE is not negligible over 5 year period. Patients with persistent endoleak and/or low platelet count should carefully be observed for LSE. Clinical Trial Registration: UMIN-CTR (UMIN000008345). PMID:27375803

  12. Myocardial longitudinal peak systolic acceleration (pSac): relationship to ejection phase, pressure, and contractility.

    PubMed

    Odland, Hans Henrik; Brun, Henrik; Sejersted, Yngve; Dalen, Marit; Edvardsen, Thor; Saugstad, Ola Didrik; Thaulow, Erik

    2012-05-01

    Acceleration has been measured both noninvasively and invasively, during both isovolumic contraction and early ejection and has been shown to reflect contractility, especially through correlation with dP/dt(max) . In this study timing and amplitude of mitral valve annulus acceleration assessed by tissue Doppler were measured and related to diastolic and systolic events. Invasive load independent measures of contractility, based on pressure-volume relationships, were derived, and pacing was done to modulate and control heart rate. Peak systolic acceleration (pSac) of the mitral valve annulus was shown to occur slightly later but timely related to dP/dt(max) (P < 0.05), while peak preejection acceleration (pPac) was related to diastolic events. During inotropy and preload modulation dP/dt(max) was found to be the strongest determinant of pSac (β= 0.9 ± 0.1; P < 0.001 and β= 1.3 ± 0.4; P < 0.001, respectively, log-transformed variables). PSac increased with pacing at a higher rate (β= 0.1 ± 0.0 cm/sec(2), P = 0.03). Furthermore, pSac indexed to end-diastolic volume (EDV) was linearly correlated to load independent contractility parameters (E(max), R = 0.7; ESPVR, R = 0.7; and PRSW, R = 0.5), and proved stable toward changes in preload and afterload. The relation between dP/dt(max)/EDV and pSac/EDV was stable throughout the study. In conclusion we found that peak systolic longitudinal acceleration (pSac) of the mitral valve annulus during aortic valve opening is strongly associated with dP/dt(max). Indexed to EDV, pSac may represent a load independent noninvasive contractility parameter. We consider pSac a candidate marker of long-axis contractility which should be viewed upon as the long-axis contribution to dP/dt(max).

  13. FSI Simulations of Pulse Wave Propagation in Human Abdominal Aortic Aneurysm: The Effects of Sac Geometry and Stiffness

    PubMed Central

    Li, Han; Lin, Kexin; Shahmirzadi, Danial

    2016-01-01

    This study aims to quantify the effects of geometry and stiffness of aneurysms on the pulse wave velocity (PWV) and propagation in fluid–solid interaction (FSI) simulations of arterial pulsatile flow. Spatiotemporal maps of both the wall displacement and fluid velocity were generated in order to obtain the pulse wave propagation through fluid and solid media, and to examine the interactions between the two waves. The results indicate that the presence of abdominal aortic aneurysm (AAA) sac and variations in the sac modulus affect the propagation of the pulse waves both qualitatively (eg, patterns of change of forward and reflective waves) and quantitatively (eg, decreasing of PWV within the sac and its increase beyond the sac as the sac stiffness increases). The sac region is particularly identified on the spatiotemporal maps with a region of disruption in the wave propagation with multiple short-traveling forward/reflected waves, which is caused by the change in boundary conditions within the saccular region. The change in sac stiffness, however, is more pronounced on the wall displacement spatiotemporal maps compared to those of fluid velocity. We conclude that the existence of the sac can be identified based on the solid and fluid pulse waves, while the sac properties can also be estimated. This study demonstrates the initial findings in numerical simulations of FSI dynamics during arterial pulsations that can be used as reference for experimental and in vivo studies. Future studies are needed to demonstrate the feasibility of the method in identifying very mild sacs, which cannot be detected from medical imaging, where the material property degradation exists under early disease initiation. PMID:27478394

  14. Primitive fitting based on the efficient multiBaySAC algorithm.

    PubMed

    Kang, Zhizhong; Li, Zhen

    2015-01-01

    Although RANSAC is proven to be robust, the original RANSAC algorithm selects hypothesis sets at random, generating numerous iterations and high computational costs because many hypothesis sets are contaminated with outliers. This paper presents a conditional sampling method, multiBaySAC (Bayes SAmple Consensus), that fuses the BaySAC algorithm with candidate model parameters statistical testing for unorganized 3D point clouds to fit multiple primitives. This paper first presents a statistical testing algorithm for a candidate model parameter histogram to detect potential primitives. As the detected initial primitives were optimized using a parallel strategy rather than a sequential one, every data point in the multiBaySAC algorithm was assigned to multiple prior inlier probabilities for initial multiple primitives. Each prior inlier probability determined the probability that a point belongs to the corresponding primitive. We then implemented in parallel a conditional sampling method: BaySAC. With each iteration of the hypothesis testing process, hypothesis sets with the highest inlier probabilities were selected and verified for the existence of multiple primitives, revealing the fitting for multiple primitives. Moreover, the updated version of the initial probability was implemented based on a memorable form of Bayes' Theorem, which describes the relationship between prior and posterior probabilities of a data point by determining whether the hypothesis set to which a data point belongs is correct. The proposed approach was tested using real and synthetic point clouds. The results show that the proposed multiBaySAC algorithm can achieve a high computational efficiency (averaging 34% higher than the efficiency of the sequential RANSAC method) and fitting accuracy (exhibiting good performance in the intersection of two primitives), whereas the sequential RANSAC framework clearly suffers from over- and under-segmentation problems. Future work will aim at further

  15. Crystal plasticity finite element analysis of deformation behaviour in SAC305 solder joint

    NASA Astrophysics Data System (ADS)

    Darbandi, Payam

    Due to the awareness of the potential health hazards associated with the toxicity of lead (Pb), actions have been taken to eliminate or reduce the use of Pb in consumer products. Among those, tin (Sn) solders have been used for the assembly of electronic systems. Anisotropy is of significant importance in all structural metals, but this characteristic is unusually strong in Sn, making Sn based solder joints one of the best examples of the influence of anisotropy. The effect of anisotropy arising from the crystal structure of tin and large grain microstructure on the microstructure and the evolution of constitutive responses of microscale SAC305 solder joints is investigated. Insights into the effects of key microstructural features and dominant plastic deformation mechanisms influencing the measured relative activity of slip systems in SAC305 are obtained from a combination of optical microscopy, orientation imaging microscopy (OIM), slip plane trace analysis and crystal plasticity finite element (CPFE) modeling. Package level SAC305 specimens were subjected to shear deformation in sequential steps and characterized using optical microscopy and OIM to identify the activity of slip systems. X-ray micro Laue diffraction and high energy monochromatic X-ray beam were employed to characterize the joint scale tensile samples to provide necessary information to be able to compare and validate the CPFE model. A CPFE model was developed that can account for relative ease of activating slip systems in SAC305 solder based upon the statistical estimation based on correlation between the critical resolved shear stress and the probability of activating various slip systems. The results from simulations show that the CPFE model developed using the statistical analysis of activity of slip system not only can satisfy the requirements associated with kinematic of plastic deformation in crystal coordinate systems (activity of slip systems) and global coordinate system (shape changes

  16. Primitive Fitting Based on the Efficient multiBaySAC Algorithm

    PubMed Central

    Kang, Zhizhong; Li, Zhen

    2015-01-01

    Although RANSAC is proven to be robust, the original RANSAC algorithm selects hypothesis sets at random, generating numerous iterations and high computational costs because many hypothesis sets are contaminated with outliers. This paper presents a conditional sampling method, multiBaySAC (Bayes SAmple Consensus), that fuses the BaySAC algorithm with candidate model parameters statistical testing for unorganized 3D point clouds to fit multiple primitives. This paper first presents a statistical testing algorithm for a candidate model parameter histogram to detect potential primitives. As the detected initial primitives were optimized using a parallel strategy rather than a sequential one, every data point in the multiBaySAC algorithm was assigned to multiple prior inlier probabilities for initial multiple primitives. Each prior inlier probability determined the probability that a point belongs to the corresponding primitive. We then implemented in parallel a conditional sampling method: BaySAC. With each iteration of the hypothesis testing process, hypothesis sets with the highest inlier probabilities were selected and verified for the existence of multiple primitives, revealing the fitting for multiple primitives. Moreover, the updated version of the initial probability was implemented based on a memorable form of Bayes’ Theorem, which describes the relationship between prior and posterior probabilities of a data point by determining whether the hypothesis set to which a data point belongs is correct. The proposed approach was tested using real and synthetic point clouds. The results show that the proposed multiBaySAC algorithm can achieve a high computational efficiency (averaging 34% higher than the efficiency of the sequential RANSAC method) and fitting accuracy (exhibiting good performance in the intersection of two primitives), whereas the sequential RANSAC framework clearly suffers from over- and under-segmentation problems. Future work will aim at further

  17. Electromigration effect on intermetallic growth and Young's modulus in SAC solder joint

    NASA Astrophysics Data System (ADS)

    Xu, Luhua; Pang, John H. L.; Ren, Fei; Tu, K. N.

    2006-12-01

    Solid-state intermetallic compound (IMC) growth behavior plays and important role in solder joint reliability of electronic packaging assemblies. The directional impact of electromigration (EM) on the growth of interfacial IMCs in Ni/SAC/Ni, Cu/SAC/Ni single BGA ball solder joint, and fine pitch ball-grid-array (FPBGA) at the anode and cathode sides is reported in this study. When the solder joint was subjected to a current density of 5,000 A/cm2 at 125°C or 150°C, IMC layer growth on the anode interface was faster than that on the cathode interface, and both were faster than isothermal aging due to the Joule heating effect. The EM affects the IMC growth rate, as well as the composition and mechanical properties. The Young’s modulus and hardness were measured by the nanoindentation continuous stiffness measurement (CSM) from planar IMC surfaces after EM exposure. Different values were observed at the anode and cathode. The energy-dispersive x-ray (EDX) line scan analysis was conducted at the interface from the cathode to anode to study the presence of species; Ni was found in the anode IMC at SAC/Cu in the Ni/SAC/Cu joint, but not detected when the current was reverse. Electron-probe microanalysis (EPMA) measurement on the Ni/SAC/Ni specimen also confirmed the polarized Ni and Cu distributions in cathode and anode IMCs, which were (Ni0.57Cu0.43)3Sn4 and (Cu0.73Ni0.27)6Sn5, respectively. Thus, the Young’s moduli of the IMC are 141 and 175 GPa, respectively.

  18. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins

    PubMed Central

    Kimberg, Daniel V.; Field, Michael; Johnson, Judith; Henderson, Antonia; Gershon, Elaine

    1971-01-01

    The effects of several prostaglandins (PG) and a highly purified preparation of cholera enterotoxin (CT) on intestinal mucosal adenyl cyclase activity and the effect of CT on intestinal mucosal cyclic 3′,5′-adenosine monophosphate concentration were determined in guinea pig and rabbit small intestine and were correlated with the effects of the same agents on ion transport. Adenyl cyclase activity, measured in a crude membrane fraction of the mucosa, was found at all levels of the small intestine with the highest activity per milligram protein in the duodenum. The prostaglandins, when added directly to the assay, increased adenyl cyclase activity; the greatest effect (2-fold increase) was obtained with PGE1 (maximal effect at 0.03 mM) and PGE2. The prostaglandins also increased short-circuit current (SCC) in isolated guinea pig ileal mucosa, with PGE1 and PGE2 again giving the greatest effects. The prior addition of theophylline (10 mM) reduced the subsequent SCC response to PGE1 and vice versa. It was concluded, therefore, that the SCC response to PGE1, like the response to theophylline, represented active Cl secretion. CT increased adenyl cyclase activity in guinea pig and rabbit ileal mucosa when preincubated with the mucosa from 1 to 2.5 hr in vitro or for 2.5 hr in vivo but not when added directly to the assay. The increments in activity caused by PGE1 and NaF were the same in CT-treated and control mucosa. Cyclic 3′,5′-AMP concentration in rabbit ileal mucosa was increased 3.5-fold after a 2 hr preincubation with CT in vitro. Phosphodiesterase activity in the crude membrane fraction of the mucosa was unaffected by either CT or PGE1. A variety of other agents including insulin, glucagon, parathormone, thyroid-stimulating hormone, L-thyroxine, thyrocalcitonin, vasopressin, and epinephrine all failed to change adenyl cyclase activity. It is concluded that CT and certain prostaglandins produce small intestinal fluid secretion by increasing mucosal adenyl

  19. A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I.

    PubMed

    Hoffmann, Steffen; Maro, Bernard; Kubiak, Jacek Z; Polanski, Zbigniew

    2011-01-01

    The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.

  20. Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

    PubMed

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-01

    The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs "chlE."

  1. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus.

    PubMed Central

    Galgiani, J N; Hewlett, E L; Friedman, R L

    1988-01-01

    Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis. PMID:2894360

  2. Gonadoblastoma and hepatoid and endometrioid-like yolk sac tumor: an update.

    PubMed

    Ulbright, Thomas M

    2014-07-01

    Dr Robert E. Scully greatly advanced our understanding of germ cell neoplasia to the extent that it is difficult to narrow the discussion of his contributions to this topic so that it can be covered in a brief article. This article accordingly focuses on some of the recent developments concerning 2 of his major contributions in this area-the gonadoblastoma (GB) and variant morphologies of yolk sac tumor. GB was defined by Dr Scully in 1953 and its features elaborated in detail by him in 1970. This neoplasm occurred in young patients who often displayed phenotypic sex ambiguities and frequently presented with primary amenorrhea. It was bilateral in 40%, and consisted of circumscribed nests of small sex cord cells and germinoma-like cells admixed with round deposits of eosinophilic, hyaline, often calcified material. These nests were set in a spindle cell gonadal stroma with Leydig-like or lutein-like cells. Because of his work we now understand that this precursor to invasive germ cell tumors occurs in patients with a specific form of disorder of sex development, namely gonadal dysgenesis, and only in those who have a particular portion of the Y chromosome, the GB locus/TSPY gene, within the gonadal tissue. An essential element to the development of GB appears to be a defect in the genetic pathway that leads to the development of Sertoli cells. Improperly formed Sertoli cells predispose to "delayed maturation" of the gonocytes of the gonad and predispose them to undergo malignant transformation. "Undifferentiated gonadal tissue" has been proposed as the precursor to the development of GB and consists of an unorganized mixture of apparently non-neoplastic germ cells, germ cells with delayed maturation, and neoplastic germ cells with sex cord cells and gonadal stroma. Two variant morphologies of yolk sac tumor were also recognized by Dr Scully. In the hepatoid variant features similar to hepatocellular carcinoma occurred, although primitive glandular foci and lack of

  3. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  4. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  5. Hypothesis: glutaminyl cyclase inhibitors decrease risks of Alzheimer's disease and related dementias.

    PubMed

    Hennekens, Charles H; Bensadon, Benjamin A; Zivin, Robert; Gaziano, J Michael

    2015-01-01

    Alzheimer's disease and related dementias (ADRD) comprise several progressive and incurable neurodegenerative disorders that some have classified as amyloidosis. With increased aging of the world's population, the prevalence of the sporadic form of ADRD, which comprises over 99% of cases, continues to rise at an alarming rate. The enormous societal burdens of ADRD already rival those of the many other major chronic diseases causing premature morbidity and mortality in the USA and worldwide such as cardiovascular disease and cancer. At present, there is an insufficient totality of evidence concerning the efficacy and safety of any pharmacologic agents to delay slow progression or reduce complications of ADRD. In this context, glutaminyl cyclase (QC) inhibitors have shown some early possible evidence of efficacy with a reassuring safety profile. To reliably test the glutaminyl cyclase (QC) and any other promising hypotheses will require cogent data from large-scale randomized trials of sufficient size and duration. PMID:26450764

  6. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

  7. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  8. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2). PMID:21173231

  9. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers. PMID:27294564

  10. New development of the yolk sac theory in diabetic embryopathy: molecular mechanism and link to structural birth defects.

    PubMed

    Dong, Daoyin; Reece, E Albert; Lin, Xue; Wu, Yanqing; AriasVillela, Natalia; Yang, Peixin

    2016-02-01

    Maternal diabetes mellitus is a significant risk factor for structural birth defects, including congenital heart defects and neural tube defects. With the rising prevalence of type 2 diabetes mellitus and obesity in women of childbearing age, diabetes mellitus-induced birth defects have become an increasingly significant public health problem. Maternal diabetes mellitus in vivo and high glucose in vitro induce yolk sac injuries by damaging the morphologic condition of cells and altering the dynamics of organelles. The yolk sac vascular system is the first system to develop during embryogenesis; therefore, it is the most sensitive to hyperglycemia. The consequences of yolk sac injuries include impairment of nutrient transportation because of vasculopathy. Although the functional relationship between yolk sac vasculopathy and structural birth defects has not yet been established, a recent study reveals that the quality of yolk sac vasculature is related inversely to embryonic malformation rates. Studies in animal models have uncovered key molecular intermediates of diabetic yolk sac vasculopathy, which include hypoxia-inducible factor-1α, apoptosis signal-regulating kinase 1, and its inhibitor thioredoxin-1, c-Jun-N-terminal kinases, nitric oxide, and nitric oxide synthase. Yolk sac vasculopathy is also associated with abnormalities in arachidonic acid and myo-inositol. Dietary supplementation with fatty acids that restore lipid levels in the yolk sac lead to a reduction in diabetes mellitus-induced malformations. Although the role of the human yolk in embryogenesis is less extensive than in rodents, nevertheless, human embryonic vasculogenesis is affected negatively by maternal diabetes mellitus. Mechanistic studies have identified potential therapeutic targets for future intervention against yolk sac vasculopathy, birth defects, and other complications associated with diabetic pregnancies.

  11. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase.

    PubMed

    Bauer, Robert J; Evans, Thomas C; Lohman, Gregory J S

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

  12. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    PubMed Central

    Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

  13. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  14. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism. PMID:22371697

  15. Vasorelaxant effect of isoliquiritigenin, a novel soluble guanylate cyclase activator, in rat aorta.

    PubMed Central

    Yu, S M; Kuo, S C

    1995-01-01

    1. The vasorelaxant activity of isoliquiritigenin, isolated from Dalbergia odorifera T, was investigated in the phenylephrine-precontracted rat aorta by measuring tension, guanylate and adenylate cyclase activities, guanosine 3':5'-cyclic monophosphate (cyclic GMP) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 2. Isoliquiritigenin concentration-dependently relaxed rat aorta contracted with phenylephrine, KCl, U-46619, endothelin and 5-hydroxytryptamine, with EC50s of 7.4 +/- 1.6, 10.5 +/- 2.3, 14.3 +/- 3.3, 11.8 +/- 2.0 and 13.6 +/- 3.7 microM, respectively. 3. Isoliquiritigenin caused endothelium-independent relaxation of phenylephrine-precontracted rat aortic rings. Neither NG-monomethyl-L-arginine (L-NMMA) (an inhibitor of the L-arginine-NO pathway) nor oxyhaemoglobin (which binds NO) modified the relaxant effect of isoliquiritigenin. The relaxant action of isoliquiritigenin also persisted in intact aorta in the presence of indomethacin or glibenclamide. However, methylene blue, an inhibitor of soluble guanylate cyclase, abolished relaxation induced by isoliquiritigenin. 4. Incubation of rat aorta with isoliquiritigenin not only increased aortic cyclic GMP content but also caused small increases in aortic cyclic AMP content, and greatly potentiated the increases in cyclic AMP observed in the presence of forskolin. The maximum increase in cyclic GMP by isoliquiritigenin was reached earlier than the increase in cyclic AMP. This result suggests that the increases in cyclic GMP caused by isoliquiritigenin might stimulate the accumulation of cyclic AMP. 5. Concentration-dependent increases in soluble guanylate cyclase activity were observed in isoliquiritigenin (1-100 microM)- or sodium nitroprusside (SNP)-treated rat aortic smooth muscle cells, while adenylate cyclase activity was unchanged in isoliquiritigenin (100 microM)-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7599926

  16. Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

    PubMed Central

    Alexander, R W; Galper, J B; Neer, E J; Smith, T W

    1982-01-01

    We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme. PMID:6289805

  17. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  18. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    PubMed

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

  19. Control of the Diadenylate Cyclase CdaS in Bacillus subtilis

    PubMed Central

    Mehne, Felix M. P.; Schröder-Tittmann, Kathrin; Eijlander, Robyn T.; Herzberg, Christina; Hewitt, Lorraine; Kaever, Volkhard; Lewis, Richard J.; Kuipers, Oscar P.; Tittmann, Kai; Stülke, Jörg

    2014-01-01

    The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination. PMID:24939848

  20. Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors.

    PubMed

    Maier, J A; Roberts, J M; Jacobs, M M

    1989-11-01

    Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction

  1. The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1.

    PubMed

    Duda, Teresa; Fik-Rymarkiewicz, Ewa; Venkataraman, Venkateswar; Krishnan, Ramalingam; Koch, Karl-Wilhelm; Sharma, Rameshwar K

    2005-05-17

    The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

  2. Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells

    PubMed Central

    Sarkar, Shayan; Jain, Sumeet; Rai, Vineeta; Sahoo, Dipak K.; Raha, Sumita; Suklabaidya, Sujit; Senapati, Shantibhusan; Rangnekar, Vivek M.; Maiti, Indu B.; Dey, Nrisingha

    2015-01-01

    The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5′ AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era. PMID:26500666

  3. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  4. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

    PubMed Central

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  5. The first structure of a bacterial diterpene cyclase: CotB2.

    PubMed

    Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

    2014-06-01

    Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

  6. Evolutionary Divergence of Sedoheptulose 7-phosphate Cyclases Leads to Several Distinct Cyclic Products

    PubMed Central

    Asamizu, Shumpei; Xie, Pengfei; Brumsted, Corey J.; Flatt, Patricia M.; Mahmud, Taifo

    2012-01-01

    Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway, and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3–1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules. PMID:22741921

  7. Cyanobacteriochrome SesA Is a Diguanylate Cyclase That Induces Cell Aggregation in Thermosynechococcus*♦

    PubMed Central

    Enomoto, Gen; Nomura, Ryouhei; Shimada, Takashi; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

    2014-01-01

    Cyanobacteria have unique photoreceptors, cyanobacteriochromes, that show diverse spectral properties to sense near-UV/visible lights. Certain cyanobacteriochromes have been shown to regulate cellular phototaxis or chromatic acclimation of photosynthetic pigments. Some cyanobacteriochromes have output domains involved in bacterial signaling using a second messenger cyclic dimeric GMP (c-di-GMP), but its role in cyanobacteria remains elusive. Here, we characterize the recombinant Tlr0924 from a thermophilic cyanobacterium Thermosynechococcus elongatus, which was expressed in a cyanobacterial system. The protein reversibly photoconverts between blue- and green-absorbing forms, which is consistent with the protein prepared from Escherichia coli, and has diguanylate cyclase activity, which is enhanced 38-fold by blue light compared with green light. Therefore, Tlr0924 is a blue light-activated diguanylate cyclase. The protein's relatively low affinity (10.5 mm) for Mg2+, which is essential for diguanylate cyclase activity, suggests that Mg2+ might also regulate c-di-GMP signaling. Finally, we show that blue light irradiation under low temperature is responsible for Thermosynechococcus vulcanus cell aggregation, which is abolished when tlr0924 is disrupted, suggesting that Tlr0924 mediates blue light-induced cell aggregation by producing c-di-GMP. Given our results, we propose the name “sesA (sessility-A)” for tlr0924. This is the first report for cyanobacteriochrome-dependent regulation of a sessile/planktonic lifestyle in cyanobacteria via c-di-GMP. PMID:25059661

  8. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  9. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    SciTech Connect

    Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie; Belrhali, Hassan; Baeyens-Volant, Danielle; Looze, Yvan; Wintjens, René

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.

  10. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    PubMed Central

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  11. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  12. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  13. Guanine-nucleotide-dependent inhibition of adenylate cyclase of rabbit heart by glucagon.

    PubMed

    Kiss, Z; Tkachuk, V A

    1984-07-16

    The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22-40%) at 0.1-0.01 microM glucagon and required the presence of 0.01-0.1 mM GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na+, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine-nucleotide-binding protein. In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine-nucleotide-dependent manner. Activation of cyclase in the presence of alcohols was maximal (30-60%) at 0.1-1.0 microM glucagon and 0.01 mM guanine nucleotides. Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine-nucleotide-binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.

  14. Lacrimal sac compression by an anterior ethmoidal mucocele presenting as a late complication of dacryocystorhinostomy

    PubMed Central

    Olaleye, Oladejo; Salleh, Shizalia; David, Don; Bickerton, Richard

    2013-01-01

    This was an unusual case of lacrimal sac compression by an anterior ethmoidal mucocele presenting as a late complication of a dacryocystorhinostomy (DCR) that was jointly managed by ophthalmic and ENT surgeons via an endoscopic approach. A 22-year-old lady presented with a 12-month history of a painless lump in her left medial canthus area and a 6-month history of left intermittent epiphora. She had a DCR when she was 15 years old with initial symptom control until recent recurrence. There were no nasal or other eye symptoms. The rest of the eye and nasendoscopic examinations were unremarkable. An MRI scan suggested a dacryocystocele; however, a further CT scan revealed a 1.6 cm cystic lesion consistent with an anterior ethmoidal mucocele compressing the lacrimal sac. An endoscopic left anterior ethmoidectomy with marsupialisation of the mucocele was performed in combination with an endoscopic DCR. She made good post-operative recovery. PMID:24964413

  15. Multi-level security for computer networking: SAC digital network approach

    SciTech Connect

    Griess, W.; Poutre, D.L.

    1983-10-01

    For telecommunications systems simultaneously handling data of different security levels, multilevel secure (MLS) operation permits maximum use of resources by automatically providing protection to users with various clearances and needs-to-know. The strategic air command (SAC) is upgrading the primary record data system used to command and control its strategic forces. The upgrade, called the SAC Digital Network (SACDIN), is designed to provide multilevel security to support users and external interfaces, with allowed accesses ranging from unclassified to top secret. SACDIN implements a security kernel based upon the Bell and Lapadula security model. This study presents an overview of the SACDIN security architecture and describes the basic message flow across the MLS network. 7 references.

  16. Air sac adenocarcinoma of the sternum in a Quaker parrot (Myiopsitta monachus).

    PubMed

    Loukopoulos, Panayiotis; Okuni, Julius Boniface; Micco, Teresa; Garcia, Jorge P; Uzal, Francisco A; Diab, Santiago S

    2014-12-01

    Respiratory neoplasia is rarely reported in avian species. A 17-yr-old Quaker parrot (Myiopsitta monachus) was admitted with a 2-wk history of anorexia, depression, and respiratory distress. Clinical examination revealed a large, firm mass in the left pectoral muscle. Radiology showed a mass silhouetting the heart and the liver. Supportive treatment was provided, but the bird died during the seventh weekly visit to drain thoracic cavity fluid. Necropsy showed a white, 3 x 3 x 2-cm, hard, gritty sternal mass. Histology showed a nonencapsulated, moderately differentiated air sac carcinoma of the sternum. Immunohistochemically the neoplasm was cytokeratin positive and vimentin and calretinin negative. This is the first report of an air sac neoplasia in a Quaker parrot and one of few respiratory tumors in psittacines.

  17. Development of SAC-OCDMA in FSO with multi-wavelength laser source

    NASA Astrophysics Data System (ADS)

    Moghaddasi, Majid; Mamdoohi, Ghazaleh; Muhammad Noor, Ahmad Shukri; Mahdi, Mohd Adzir; Ahmad Anas, Siti Barirah

    2015-12-01

    We propose and demonstrate a free space optical network, based on spectral amplitude coding optical code division multiple access (SAC-OCDMA) with a multi-wavelength laser source. A detailed theoretical analysis that represents the characteristics of SAC-OCDMA system was developed. In addition to the impact of turbulence, influences of several system noises such as optical beat interference (OBI), relative intensity noise, and receiver noises, have been studied. From the numerical results, it was found that the influence of OBI is more dominant, especially at higher received power. Two different codes, namely, modified quadratic congruence and modified double weight, are then compared with the latter which provides better performance. A transmission distance of 2.6 km with 10 users and an 8 cm aperture diameter is advisable whenever the turbulence is moderate. These results can be improved when a beam divergence smaller than 1 mrad is utilized.

  18. Multi-level security for computer networking - SAC digital network approach

    NASA Astrophysics Data System (ADS)

    Griess, W.; Poutre, D. L.

    The functional features and architecture of the SACDIN (SAC digital network) are detailed. SACDIN is the new data transmission segment for directing SAC's strategic forces. The system has 135 processor nodes at 32 locations and processes, distributes and stores data of any level of security classification. The sophistication of access nodes is dependent on the location. A reference monitor mediates the multilevel security by implementation of the multi-state machine concept, i.e., the Bell-LaPadula model (1973, 1974), which concludes that a secure state can never lead to an unsecure state. The monitor is controlled by the internal access control mechanism, which resides in PROM. Details of the access process are provided, including message flow on trusted paths appropriate to the security clearance of the user.

  19. A Rare Case of Endolymphatic Sac Tumour: Clinicopathologic Study and Surgical Management

    PubMed Central

    Ferri, Emanuele; Amadori, Maurizio; Armato, Enrico; Pavon, Ida

    2014-01-01

    Objective. Endolymphatic sac tumor (ELST) is a rare neoplasm arising from the intrapetrous portion of the endolymphatic sac, either isolated or in association with the von Hippel-Lindau disease. We report a sporadic case of ELST with an overview of the literature and a discussion of clinic-radiological, histopathologic, and surgical findings. Case Report. A young woman presented with a progressive hearing loss in the left ear. Otoscopy showed a reddish, bleeding hypotympanic mass. CT demonstrated an expansile lytic mastoid lesion extending to the middle ear, with bone erosion. MRI confirmed a lesion of increased signal on T1-weighted sequences. The patient underwent a canal wall-down tympanoplasty with complete removal of the tumor. Histopathology was consistent with a papillary ELST. Immunohistochemistry was positive for cytokeratin and chromogranin A. Conclusion. This paper highlights the rarity of ELST, the need for an accurate neuroradiological and immunohistochemical study at the early stages, and the timeliness of surgical treatment. PMID:24991442

  20. Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB).

    PubMed

    Méndez-Lorenzo, Luz; Porras-Domínguez, Jaime R; Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín

    2015-01-01

    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final

  1. Amines in the marking fluid and anal sac secretion of the tiger, Panthera tigris.

    PubMed

    Banks, G R; Buglass, A J; Waterhouse, J S

    1992-01-01

    Analysis of the marking fluid of two tigers (one Bengal and one Sumatran) by GC using an amine-specific column and a nitrogen-specific detector has shown the presence of the following amines: ammonia, methylamine, dimethylamine, trimethylamine, triethylamine, propylamine, and butane-1,4-diamine (putrescine). In contrast to previously published reports, we were unable to detect 2-phenylethylamine. The anal sac secretion was found to have a similar amine content.

  2. KNApSAcK Metabolite Activity Database for retrieving the relationships between metabolites and biological activities.

    PubMed

    Nakamura, Yukiko; Afendi, Farit Mochamad; Parvin, Aziza Kawsar; Ono, Naoaki; Tanaka, Ken; Hirai Morita, Aki; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

    2014-01-01

    Databases (DBs) are required by various omics fields because the volume of molecular biology data is increasing rapidly. In this study, we provide instructions for users and describe the current status of our metabolite activity DB. To facilitate a comprehensive understanding of the interactions between the metabolites of organisms and the chemical-level contribution of metabolites to human health, we constructed a metabolite activity DB known as the KNApSAcK Metabolite Activity DB. It comprises 9,584 triplet relationships (metabolite-biological activity-target species), including 2,356 metabolites, 140 activity categories, 2,963 specific descriptions of biological activities and 778 target species. Approximately 46% of the activities described in the DB are related to chemical ecology, most of which are attributed to antimicrobial agents and plant growth regulators. The majority of the metabolites with antimicrobial activities are flavonoids and phenylpropanoids. The metabolites with plant growth regulatory effects include plant hormones. Over half of the DB contents are related to human health care and medicine. The five largest groups are toxins, anticancer agents, nervous system agents, cardiovascular agents and non-therapeutic agents, such as flavors and fragrances. The KNApSAcK Metabolite Activity DB is integrated within the KNApSAcK Family DBs to facilitate further systematized research in various omics fields, especially metabolomics, nutrigenomics and foodomics. The KNApSAcK Metabolite Activity DB could also be utilized for developing novel drugs and materials, as well as for identifying viable drug resources and other useful compounds.

  3. Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

    PubMed Central

    Raga-Carbajal, Enrique; Olvera, Clarita; Rodríguez-Alegría, Maria Elena; Carrillo-Nava, Ernesto; Costas, Miguel; López Munguía, Agustín

    2015-01-01

    Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final

  4. Amines in the marking fluid and anal sac secretion of the tiger, Panthera tigris.

    PubMed

    Banks, G R; Buglass, A J; Waterhouse, J S

    1992-01-01

    Analysis of the marking fluid of two tigers (one Bengal and one Sumatran) by GC using an amine-specific column and a nitrogen-specific detector has shown the presence of the following amines: ammonia, methylamine, dimethylamine, trimethylamine, triethylamine, propylamine, and butane-1,4-diamine (putrescine). In contrast to previously published reports, we were unable to detect 2-phenylethylamine. The anal sac secretion was found to have a similar amine content. PMID:1388518

  5. Computed tomographic epidurography: an aid to understanding deformation of the lumbar dural sac by epidural injections.

    PubMed

    Fukushige, T; Kano, T; Sano, T; Irie, M

    1999-09-01

    Local anaesthetics injected into the epidural space may deform the dural sac to a variable degree, thereby contributing to variability in the extent of the block. We investigated deformation of the lumbar dural sac after injection into the lumbar epidural space. The subjects were 26 patients with low-back pain who underwent lumbar epidurography and computed tomographic (CT) epidurography, of whom seven also underwent myelography and computed tomographic myelography. The epidural space was entered via the sacral hiatus in 24 patients and through the L5/S1 interspace in two patients. Ten millilitres of local anaesthetic was then injected into the epidural space followed by 20 mL of contrast medium. Computed tomographic epidurography was undertaken approximately 30-min after the epidural injection at the mid-vertebral and mid-discal levels from the first lumbar through to the first sacral vertebrae. The dural sac usually showed an oval or hexagonal shape on the transverse views at the first and second lumbar vertebral levels, and the shape of an inverted triangle below the level of the third lumbar vertebra. A median line of translucency was also observed on the posteroanterior epidurographic view in 25 of the 26 patients. This line was though to be a manifestation of the dural deformation to the inverted triangle. Dural sac deformation usually shows a specific pattern, although there are individual variations. Dural deformability is an important consideration in any analysis of the spread of epidural block or of the changes of epidural pressure after epidural injection of local anaesthetics. PMID:10549463

  6. Silver(I)-saccharin coordination polymers with 2-pyridylmethanol and 2,6-pyridinedimethanol. Synthetic, spectroscopic, thermal and structural studies of [Ag(sac)( μ-mpy)] n and [Ag( μ-sac)(dmpy)] n

    NASA Astrophysics Data System (ADS)

    Hamamci, Sevim; Yilmaz, Veysel T.; Harrison, William T. A.; Thöne, Carsten

    2005-04-01

    Two new coordination polymers of saccharinato-silver(I) complexes with 2-pyridylmethanol (mpy) and 2,6-pyridinedimethanol (dmpy), [Ag(sac)( μ-mpy)] n ( 1) and [Ag( μ-sac)(dmpy)] n ( 2), (sac = saccharinate), have been synthesized and structurally characterized by elemental analysis, IR, thermal analysis and single crystal X-ray diffraction. Crystal data: 1, monoclinic, P2/c, with a=13.3584(14) Å, b=8.3040(10) Å, c=13.0430(14) Å, β=106.227(3)°, Z=4; 2, monoclinic, P2/n, with a=7.3538(1) Å, b=13.0444(3) Å, c=15.7315(3) Å, β=98.8976(13)°, Z=4. In 1, [Ag 2(sac) 2(mpy) 2] building blocks are connected into a two-dimensional framework by bridging OH groups of mpy, and they are further stacked by O sbnd H⋯O hydrogen bonds, and weak π(sac)⋯ π(py) and π(py)⋯ π(py) interactions to form a three-dimensional supramolecular framework. In 2, [Ag 2(sac)(dmpy)] units are linked by the O sulfonyl bridges of sac, leading to one-dimensional polymeric chains, which are reinforced by the O sbnd H⋯O hydrogen bonds and π(sac)⋯ π(py) interactions to generate a three-dimensional supramolecular network. The coordination polymers 1 and 2 exhibit three- and four- coordinated silver(I) centers, respectively, and the intra-chain Ag⋯Ag separations in 1 and 2 are 3.2988(4) and 4.1429(2) Å, respectively.

  7. Characterization of calcium carbonate crystals in pigeon yolk sacs with different incubation times.

    PubMed

    Song, Juan; Cheng, Haixia; Shen, Xinyu; Hu, Jingxiao; Tong, Hua

    2014-05-01

    Calcium carbonate crystals are known to form in the yolk sacs of fertile pigeon eggs at late stages of incubation. The composition and structure of these crystals were investigated, the crystallization environment was inspected, and the physical chemistry constants of the yolk fluid were determined through the incubation period. Polarized light microscopy was used to observe the generation and distribution of calcium carbonate crystals in the yolk sac. In addition, X-ray diffraction was employed to analyze the composition and crystal phase of the yolk sac. A decalcification and deproteination method was established to analyze the ultrastructure and composition of the crystals, as well as the internal relationship between inorganic and organic phases of the crystals. Additionally, scanning electron microscopy, transmission electron microscopy, X-ray energy dispersive spectroscopy, and Fourier transform infrared spectroscopy were used to evaluate the characteristics of the crystals. Our results demonstrated that the calcium carbonate crystals were mainly composed of vaterite and calcite, with vaterite being the major component. Vaterite, a type of biomaterial generated by an organic template control, presented as a concentric hierarchical spherical structure. The organic nature of the biomaterial prevented vaterite from transforming into calcite, which is more thermodynamically stable than vaterite. Additionally, the configuration, size, and aggregation of vaterite were also mediated by the organic template. This bio-vaterite was found during the incubation period and is valuable in calcium transport during embryonic development.

  8. Pathological, immunohistochemical and microbiologicalal analysis of lacrimal sac biopsies in patients with chronic dacrocystitis

    PubMed Central

    Amin, Rowayda Mahmoud; Hussein, Faten Aly; Idriss, Hisham Farouk; Hanafy, Nesrine Fathy; Abdallah, Dina Mohamed

    2013-01-01

    AIM To analyze cases of obstruction of the nasolacrimal duct which creates a fertile environment for secondary bacterial infection and can result in dacryocystitis, which is a constant threat to cornea and orbital soft tissue and a potential source of endophthalmitis following intraocular surgery. The majority of obstructions of the lacrimal excretory outflow system are acquired ones occurring in adulthood and involving the distal parts of the system. Acquired obstruction may be primary/idiopathic or secondary to a wide variety of infectious, inflammatory, traumatic, mechanical, toxic or neoplastic causes mimicking idiopathic inflammation. These cases are treated by dacryocystorhinostomy (DCR). METHODS The present study was conducted to determine the histopathologic, immunohistochemical and current microbiologic characteristics of lacrimal sac specimens in patients undergoing external dacryocystorhinostomy. RESULTS Non-specific lacrimal sac pathology was present in all 33 cases and 81.8% of the cases showed moderate chronic inflammation with a chronic inflammatory score (CIS) ranging between 4 and 6, whereas 12.12% showed severe inflammatory changes with a CIS of 7. Mild degree of inflammation was seen in 6.06% with a CIS of 3. The total prevalence of gram-positive, gram-negative, and culture-negative samples were 59.4%, 37.5%, and 3% respectively. CONCLUSION Non-specific chronic inflammation with fibrosis is indeed the most commonly reported histopathological finding in lacrimal sac wall biopsy specimens. PMID:24392331

  9. Evaluation of Refractivity Profiles from CHAMP and SAC-C GPS Radio Occultation

    NASA Technical Reports Server (NTRS)

    Poli, Paul; Ao, Chi On; Joiner, Joanna; delaTorreJuarez, Manuel; Hoff, Raymond

    2002-01-01

    The GeoForschungsZentrum's Challenging Minisatellite Payload for Geophysical Research and Application (CHAMP, Germany-US) and the Comision Nacional de Actividades Especiales' Satelite de Aplicaciones Cientificas-C (SAC-C, Argentina-US) missions are the first missions to carry a second-generation Blackjack Global Positioning System (GPS) receiver. One of the new features of this receiver is its ability to sense the lower troposphere closer to the surface than the proof-of-concept GPS Meteorology experiment (GPS/MET). Since their launch, CHAMP and SAC-C have collected thousands of GPS radio occultations, representing a wealth of measurements available for data assimilation and Numerical Weather Prediction (NWP). In order to evaluate the refractivity data derived by the Jet Propulsion Laboratory (JPL) from raw radio occultation measurements, we use Data Assimilation Office (DAO) 6-hour forecasts as an independent state of the atmosphere. We compare CHAMP and SAC-C refractivity (processed by JPL) with refractivity calculated from the DAO global fields of temperature, water vapor content and humidity. We show statistics of the differences as well as histograms of the differences.

  10. The sac Mutants of Chlamydomonas reinhardtii Reveal Transcriptional and Posttranscriptional Control of Cysteine Biosynthesis1

    PubMed Central

    Ravina, Cristina G.; Chang, Chwenn-In; Tsakraklides, George P.; McDermott, Jeffery P.; Vega, Jose M.; Leustek, Thomas; Gotor, Cecilia; Davies, John P.

    2002-01-01

    Algae and vascular plants are cysteine (Cys) prototrophs. They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds. Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway. The derived amino acid sequences of the C. reinhardtii genes encoding 5′-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants. The Cys biosynthetic pathway of C. reinhardtii is regulated by sulfate availability. The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate. The sac1 mutation, which impairs C. reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase. The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity. These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process. PMID:12481091

  11. Heterogeneity of vocal sac inflation patterns in Odorrana tormota plays a role in call diversity.

    PubMed

    Zhang, Fang; Zhao, Juan; Chen, Pan; Chen, Zhuqing; Chen, Yuanyuan; Feng, Albert S

    2016-03-01

    Male concave-eared torrent frogs (Odorrana tormota) can emit at least eight distinct call-types. However, the mechanisms by which they are produced are not fully understood. Anatomical analysis revealed that the vocal sacs of male O. tormota comprise two physically distinct compartments (pars lateralis and pars ventralis), residing on two sides of the vocal slits. The goal of the present study was to test the hypothesis that the two compartments play a role in the production of the diverse call-types. For this, audio and video recordings of male vocalizations were made, and sounds were analyzed afterwards. Results showed that the vocal sac inflation pattern was heterogeneous, and the call duration was a major factor determining the differential inflation patterns. Short call-types (duration <200 ms) involved inflation of one of the compartments only; those having a fundamental frequency (F0) of >5000 Hz involved inflation of pars lateralis only, whereas those with an F0 of <4000 Hz (tone-pips and "infant" calls) involved inflation of pars ventralis only. Long call-types (duration >200 ms), e.g., shallow frequency modulation calls, staccato calls, and long calls, involved inflation of both compartments of the vocal sacs. These results give support to the working hypothesis.

  12. A case of congenital diaphragmatic hernia with a hernia sac attached to the liver: hints for an early embryological insult.

    PubMed

    Sharma, Shilpa; Jain, Rani; Singh, M K; Gupta, Devendra K

    2007-10-01

    We describe here the unusual operative findings in a case of congenital diaphragmatic hernia (CDH). A neonate antenatally diagnosed as having CDH was operated via a left subcostal incision. The operative findings included a large central diaphragmatic defect of 5 x 5 cm, lined by a thick sac. The contents of the hernia included the stomach, spleen, and loops of the small and large intestine. The left side of the liver was thinned out and forming a part of the dome of the sac of the CDH. Vessels of the hepatic tissue were in continuity with the sac. There were scattered liver tissues forming cords in the sac lining. These findings were confirmed by histopathologic examinations. These findings have not been reported earlier in humans and might help to elucidate the embryology of the development of the diaphragm. Our findings suggest that this kind of defect occurs early in development, and we hypothesize that it is associated with a poorer prognosis.

  13. Immunohistochemical expression of SALL4 in hepatocellular carcinoma, a potential pitfall in the differential diagnosis of yolk sac tumors.

    PubMed

    Gonzalez-Roibon, Nilda; Katz, Betina; Chaux, Alcides; Sharma, Rajni; Munari, Enrico; Faraj, Sheila F; Illei, Peter B; Torbenson, Michael; Netto, George J

    2013-07-01

    SALL4 is a transcription factor that serves as a marker of yolk sac tumor. Yolk sac tumor and hepatocellular carcinoma share histologic, serologic, and immunohistochemical features. Previous studies have shown lack of SALL4 expression in hepatocellular carcinoma, suggesting utility in this differential diagnosis. Sixty-nine samples of hepatocellular carcinoma were retrieved from surgical pathology archives and used to construct 9 tissue microarrays. A germ cell tumor tissue microarray containing 10 yolk sac tumors was used for comparison. Extent, intensity, and pattern of nuclear SALL4 expression were assessed in each spot. Mean percentage of expression was calculated for each tumor and used during analysis. Optimal discriminatory extent of expression cutoff was determined by receiver operating characteristic curve analysis. Other potential discriminatory markers including Hep Par1 were also evaluated. Forty-six percent (32/69) of hepatocellular carcinoma and all yolk sac tumors revealed at least focal expression of SALL4. A unique punctuate/clumped pattern of nuclear staining was present in 94% (30/32) of hepatocellular carcinoma, whereas all yolk sac tumors displayed a diffuse finely granular nuclear staining pattern. A 25% extent of SALL4 expression cutoff was found to be optimal for the distinction of yolk sac tumor from hepatocellular carcinoma yielding a sensitivity of 100%, specificity of 92.8%, and a positive predictive value of 66.6% for yolk sac tumor diagnosis. The addition of Hep Par1 increased the specificity (99%) and positive predictive value (90%). This is the first report of SALL4 expression in hepatocellular carcinoma. Our finding should be taken into consideration in the differential diagnosis of hepatocellular carcinoma and yolk sac tumor. The unique punctuate/clumped pattern seen in hepatocellular carcinoma cases could be of further discriminatory value. PMID:23347651

  14. Ultrastructure of the endolymphatic sac in the larva of the japanese red-bellied newt Cynops pyrrhogaster

    NASA Technical Reports Server (NTRS)

    Gao, W.; Wiederhold, M.; Hejl, R.

    1998-01-01

    The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the "floccular" vesicle and the "granular" vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain floccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.

  15. Physiological requirements for 20-hydroxyecdysone-induced rectal sac distention in the pupa of the silkworm, Bombyx mori.

    PubMed

    Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

    2010-06-01

    Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention.

  16. Transcript profiles of maize embryo sacs and preliminary identification of genes involved in the embryo sac–pollen tube interaction

    PubMed Central

    Wang, Shuai Shuai; Wang, Fang; Tan, Su Jian; Wang, Ming Xiu; Sui, Na; Zhang, Xian Sheng

    2014-01-01

    The embryo sac, the female gametophyte of flowering plants, plays important roles in the pollination and fertilization process. Maize (Zea mays L.) is a model monocot, but little is known about the interactions between its embryo sac and the pollen tube. In this study, we compared the transcript profiles of mature embryo sacs, mature embryo sacs 14–16 h after pollination, and mature nucelli. Comparing the transcript profiles of the embryo sacs before and after the entry of the pollen tube, we identified 3467 differentially expressed transcripts (3382 differentially expressed genes; DEGs). The DEGs were grouped into 22 functional categories. Among the DEGs, 221 genes were induced upon the entry of the pollen tube, and many of them encoded proteins involved in RNA binding, processing, and transcription, signaling, miscellaneous enzyme family processes, and lipid metabolism processes. Genes in the DEG dataset were grouped into 17 classes in a gene ontology enrichment analysis. The DEGs included many genes encoding proteins involved in protein amino acid phosphorylation and protein ubiquitination, implying that these processes might play important roles in the embryo sac–pollen tube interaction. Additionally, our analyses indicate that the expression of 112 genes encoding cysteine-rich proteins (CRPs) is induced during pollination and fertilization. The CRPs likely regulate pollen tube guidance and embryo sac development. These results provide important information on the genes involved in the embryo sac–pollen tube interaction in maize. PMID:25566277

  17. Influencing Factors for Abdominal Aortic Aneurysm Sac Shrinkage and Enlargement after EVAR: Clinical Reviews before Introduction of Preoperative Coil Embolization

    PubMed Central

    Hiraoka, Arudo; Totsugawa, Toshinori; Tamura, Kentaro; Ishida, Atsuhisa; Sakaguchi, Taichi; Yoshitaka, Hidenori

    2014-01-01

    Background: We previously reported effectiveness of coil embolization (CE) to aortic branched vessels before endovascular aortic repair (EVAR) for abdominal aortic aneurysm (AAA) because of significant shrinkage of aneurysmal sac. In this study, we investigated EVAR cases to clarify influential factors of aneurysmal shrinkage and enlargement. Methods: 148 consecutive cases before the introduction of CE were retrospectively reviewed based on the presence of PT2EL (persistent type 2 endoleak) and change in sac diameter after EVAR by multivariate analysis. Results: (A) PT2EL risk factors were patent inferior mesenteric artery (IMA) and thinner mural thrombus inside aneurysmal sac. (B) Sac enlargement risk factors were antiplatelet intake, PT2EL, and female gender. (C) Sac shrinkage predictive factors were the absences of thoracic aortic aneurysm, antiplatelet intake, PT2EL, and coronary artery disease. Conclusion: CE to IMA was considered to be effective because patent IMA and antiplatelet intake were significant risk factors for sac enlargement. So, more meticulous therapeutic strategy, including treatment priority (AAA first or CAD first) and choice of treatment (EVAR vs. AAA) based on anatomical features of AAA was required to improve late outcomes. PMID:25298830

  18. β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

    PubMed

    Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

    2016-06-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552.

  19. Penguin lungs and air sacs: implications for baroprotection, oxygen stores and buoyancy.

    PubMed

    Ponganis, P J; St Leger, J; Scadeng, M

    2015-03-01

    The anatomy and volume of the penguin respiratory system contribute significantly to pulmonary baroprotection, the body O2 store, buoyancy and hence the overall diving physiology of penguins. Therefore, three-dimensional reconstructions from computerized tomographic (CT) scans of live penguins were utilized to measure lung volumes, air sac volumes, tracheobronchial volumes and total body volumes at different inflation pressures in three species with different dive capacities [Adélie (Pygoscelis adeliae), king (Aptenodytes patagonicus) and emperor (A. forsteri) penguins]. Lung volumes scaled to body mass according to published avian allometrics. Air sac volumes at 30 cm H2O (2.94 kPa) inflation pressure, the assumed maximum volume possible prior to deep dives, were two to three times allometric air sac predictions and also two to three times previously determined end-of-dive total air volumes. Although it is unknown whether penguins inhale to such high volumes prior to dives, these values were supported by (a) body density/buoyancy calculations, (b) prior air volume measurements in free-diving ducks and (c) previous suggestions that penguins may exhale air prior to the final portions of deep dives. Based upon air capillary volumes, parabronchial volumes and tracheobronchial volumes estimated from the measured lung/airway volumes and the only available morphometry study of a penguin lung, the presumed maximum air sac volumes resulted in air sac volume to air capillary/parabronchial/tracheobronchial volume ratios that were not large enough to prevent barotrauma to the non-collapsing, rigid air capillaries during the deepest dives of all three species, and during many routine dives of king and emperor penguins. We conclude that volume reduction of airways and lung air spaces, via compression, constriction or blood engorgement, must occur to provide pulmonary baroprotection at depth. It is also possible that relative air capillary and parabronchial volumes are

  20. Penguin lungs and air sacs: implications for baroprotection, oxygen stores and buoyancy.

    PubMed

    Ponganis, P J; St Leger, J; Scadeng, M

    2015-03-01

    The anatomy and volume of the penguin respiratory system contribute significantly to pulmonary baroprotection, the body O2 store, buoyancy and hence the overall diving physiology of penguins. Therefore, three-dimensional reconstructions from computerized tomographic (CT) scans of live penguins were utilized to measure lung volumes, air sac volumes, tracheobronchial volumes and total body volumes at different inflation pressures in three species with different dive capacities [Adélie (Pygoscelis adeliae), king (Aptenodytes patagonicus) and emperor (A. forsteri) penguins]. Lung volumes scaled to body mass according to published avian allometrics. Air sac volumes at 30 cm H2O (2.94 kPa) inflation pressure, the assumed maximum volume possible prior to deep dives, were two to three times allometric air sac predictions and also two to three times previously determined end-of-dive total air volumes. Although it is unknown whether penguins inhale to such high volumes prior to dives, these values were supported by (a) body density/buoyancy calculations, (b) prior air volume measurements in free-diving ducks and (c) previous suggestions that penguins may exhale air prior to the final portions of deep dives. Based upon air capillary volumes, parabronchial volumes and tracheobronchial volumes estimated from the measured lung/airway volumes and the only available morphometry study of a penguin lung, the presumed maximum air sac volumes resulted in air sac volume to air capillary/parabronchial/tracheobronchial volume ratios that were not large enough to prevent barotrauma to the non-collapsing, rigid air capillaries during the deepest dives of all three species, and during many routine dives of king and emperor penguins. We conclude that volume reduction of airways and lung air spaces, via compression, constriction or blood engorgement, must occur to provide pulmonary baroprotection at depth. It is also possible that relative air capillary and parabronchial volumes are

  1. Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

    PubMed Central

    Charoonroje, P; Tokumitsu, Y; Nomura, Y

    1994-01-01

    1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system. PMID:7834209

  2. Desensitization of the guinea-pig urinary bladder by the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate and by substance P.

    PubMed Central

    Hourani, S. M.

    1984-01-01

    Adenosine 5'-triphosphate (ATP), substance P (SP) and non-cholinergic nerve stimulation contracted the guinea-pig urinary bladder. SP and two poorly-degradable analogues of ATP, the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate (AMP-PCP and L-AMP-PCP), were used to desensitize the guinea-pig bladder. Desensitization of the bladder by AMP-PCP (50 microM) or by L-AMP-PCP (50 microM) abolished the responses to ATP, and inhibited the responses to non-cholinergic nerve stimulation and to SP. The responses to histamine were unaffected. Desensitization by SP (1 microM) inhibited the responses to SP itself, but not the responses to ATP, L-AMP-PCP or non-cholinergic nerve stimulation. These results suggest that SP may act partly by releasing ATP, and support the suggestion that ATP rather than SP is the non-cholinergic stimulatory transmitter. PMID:6203588

  3. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. PMID:27214242

  4. Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

    PubMed Central

    2015-01-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate. PMID:24832673

  5. Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

    PubMed Central

    Lopez, Miguel A.; Saada, Edwin A.

    2014-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  6. Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins

    PubMed Central

    Iyer, Lakshminarayan M; Anantharaman, Vivek; Aravind, L

    2003-01-01

    Background Soluble guanylyl cyclases (SGCs) are dimeric enzymes that transduce signals downstream of nitric oxide (NO) in animals. They sense NO by means of a heme moiety that is bound to their N-terminal extensions. Results Using sequence profile searches we show that the N-terminal extensions of the SGCs contain two globular domains. The first of these, the HNOB (Heme NO Binding) domain, is a predominantly α-helical domain and binds heme via a covalent linkage to histidine. Versions lacking this conserved histidine and are likely to interact with heme non-covalently. We detected HNOB domains in several bacterial lineages, where they occur fused to methyl accepting domains of chemotaxis receptors or as standalone proteins. The standalone forms are encoded by predicted operons that also contain genes for two component signaling systems and GGDEF-type nucleotide cyclases. The second domain, the HNOB associated (HNOBA) domain occurs between the HNOB and the cyclase domains in the animal SGCs. The HNOBA domain is also detected in bacteria and is always encoded by a gene, which occurs in the neighborhood of a gene for a HNOB domain. Conclusion The HNOB domain is predicted to function as a heme-dependent sensor for gaseous ligands, and transduce diverse downstream signals, in both bacteria and animals. The HNOBA domain functionally interacts with the HNOB domain, and possibly binds a ligand, either in cooperation, or independently of the latter domain. Phyletic profiles and phylogenetic analysis suggest that the HNOB and HNOBA domains were acquired by the animal lineage via lateral transfer from a bacterial source. PMID:12590654

  7. Structure of a sedoheptulose 7-phosphate cyclase: ValA from Streptomyces hygroscopicus.

    PubMed

    Kean, Kelsey M; Codding, Sara J; Asamizu, Shumpei; Mahmud, Taifo; Karplus, P Andrew

    2014-07-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp→Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate.

  8. Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

    PubMed

    Lopez, Miguel A; Saada, Edwin A; Hill, Kent L

    2015-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed "social motility," based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  9. Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

    PubMed Central

    Marumo, Fumiaki; Edelman, Isidore S.

    1971-01-01

    Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed. PMID:4329002

  10. Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments

    PubMed Central

    1989-01-01

    Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition. In tROS, exogenously applied GTP produced a dark current whose amplitude was half-maximal at approximately 0.4 mM GTP. The conductance for this current was suppressed by light in a fashion similar to when it is activated by cGMP. In addition, no current was produced in the absence of Mg2+, which is known to be necessary for the guanylate cyclase activity. These results indicate that guanylate cyclase was present in tROS and synthesized cGMP from exogenously applied GTP. The enzyme activity was distributed throughout the rod outer segment. The amount of synthesized cGMP increased as the cytoplasmic Ca2+ concentration of tROS decreased, which indicated the activation of guanylate cyclase at low Ca2+ concentrations. Half-maximal effect of Ca2+ was observed at approximately 100 nM. tROS contained the proteins involved in the phototransduction mechanism and therefore, we could examine the regulation of the light response waveform by Ca2+. At low Ca2+ concentrations, the time course of the light response was speeded up probably because cGMP recovery was facilitated by activation of the cyclase. Then, if the cytoplasmic Ca2+ concentration of a photoreceptor decreases during light stimulation, the Ca2+ decrease may explain the acceleration of the light response during light adaptation. In tROS, however, we did observe an acceleration during repetitive light flashes when the cytoplasmic Ca2+ concentration increased during the stimulation. This result suggests the

  11. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

    PubMed Central

    Sternweis, P C; Gilman, A G

    1982-01-01

    Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-. PMID:6289322

  12. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    SciTech Connect

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-06-24

    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  13. The acid-induced folded state of Sac7d is the native state.

    PubMed Central

    Bedell, J. L.; McCrary, B. S.; Edmondson, S. P.; Shriver, J. W.

    2000-01-01

    Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid. PMID:11106160

  14. Yolk sac mesenchymal progenitor cells from New World mice (Necromys lasiurus) with multipotent differential potential.

    PubMed

    Favaron, Phelipe Oliveira; Mess, Andrea; Will, Sônia Elisabete; Maiorka, Paulo César; de Oliveira, Moacir Franco; Miglino, Maria Angelica

    2014-01-01

    Fetal membranes are abundant, ethically acceptable and readily accessible sources of stem cells. In particular, the yolk sac is a source of cell lineages that do not express MHCs and are mainly free from immunological incompatibles when transferred to a recipient. Although data are available especially for hematopoietic stem cells in mice and human, whereas other cell types and species are dramatically underrepresented. Here we studied the nature and differentiation potential of yolk sac derived mesenchymal stem cells from a New World mouse, Necromys lasiurus. Explants from mid-gestation were cultured in DMEM-High glucose medium with 10% defined fetal bovine serum. The cells were characterized by standard methods including immunophenotyping by fluorescence and flow cytometry, growth and differentiation potential and tumorigenicity assays. The first adherent cells were observed after 7 days of cell culture and included small, elongated fibroblast-like cells (92.13%) and large, round epithelial-like cells with centrally located nuclei (6.5%). Only the fibroblast-like cells survived the first passages. They were positive to markers for mesenchymal stem cells (Stro-1, CD90, CD105, CD73) and pluripotency (Oct3/4, Nanog) as well as precursors of hematopoietic stem cells (CD117). In differentiation assays, they were classified as a multipotent lineage, because they differentiated into osteogenic, adipogenic, and chondrogenic lineages and, finally, they did not develop tumors. In conclusion, mesenchymal progenitor cells with multipotent differentiation potential and sufficient growth and proliferation abilities were able to be obtained from Necromys yolk sacs, therefore, we inferred that these cells may be promising for a wide range of applications in regenerative medicine.

  15. [Sigmoidoperianal fistula associated with diverticulitis and Cul de Sac situation--diagnostics and therapy].

    PubMed

    Weyand, G; Rinast, E; Englert, A; Houf, M

    2002-07-01

    We report on the case of a 64-year-old female patient who presented herself in our outpatient clinic because of a perianal fistula with recurrent abscesses. We describe the step diagnostics and the surgical treatment of the causal sigmoido-perianal fistula with diverticulitis and Cul de sac situation. Clinical examination, fistulography, colonoscopy and MRT were part of the precise representation and preparation for the high anterior rectosigmoidal resection with simultaneous rectopexy according to Sudeck which were performed without complications. The sigmoidoperianal fistula must be taken into account as a differential diagnosis of a recalcitrant high perianal fistula. PMID:12122593

  16. Water quality in the Little Sac River basin near Springfield, Missouri, 1999-2001

    USGS Publications Warehouse

    Smith, Brenda J.

    2002-01-01

    The Little Sac River, north of Springfield, Missouri, flows through mainly agricultural and forest land. However, the quality of the river water is a concern because the river flows into Stockton Lake, which is a supplemental drinking water source for Springfield. Large bacterial densities and nutrient concentrations are primary concerns to the water quality of the river.A 29-river mile reach of the Little Sac River is on the 1998 list of waters of Missouri designated under section 303(d) of the Federal Clean Water Act because of fecal coliform densities larger than the Missouri Department of Natural Resources standard (hereinafter referred to as Missouri standard) of 200 colonies per 100 milliliters for whole-body contact recreation. During an investigation of the water quality in the Little Sac River by the U.S. Geological Survey, in cooperation with the Watershed Committee of the Ozarks, fecal coliform bacteria densities exceeded the Missouri standard (the standard applies from April 1 through October 31) in one sample from a site near Walnut Grove. At other sites on the Little Sac River, the Missouri standard was exceeded in two samples and equalled in one sample upstream from the Northwest Wastewater Treatment Plant (NW WTP) and in one sample immediately downstream from the NW WTP.Effluent from the NW WTP flows into the Little Sac River. Annually from April 1 through October 31, the effluent is disinfected to meet the Missouri standard for whole-body contact recreation. Fecal coliform bacteria densities in samples collected during this period generally were less than 100 colonies per 100 milliliters. For the rest of the year when the effluent was not disinfected, the bacteria densities in samples ranged from 50 (sample collected on November 1, 2000) to 10,100 colonies per 100 milliliters (both counts were non-ideal). When the effluent was disinfected and the fecal coliform bacteria density was small, samples from sites upstream and downstream from the NW WTP

  17. Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.

    PubMed

    Wallingford, Mary C; Giachelli, Cecilia M

    2014-08-01

    PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.

  18. Early sac shrinkage predicts a low risk of late complications after endovascular aortic aneurysm repair

    PubMed Central

    Bastos Gonçalves, F; Baderkhan, H; Verhagen, H J M; Wanhainen, A; Björck, M; Stolker, R J; Hoeks, S E; Mani, K

    2014-01-01

    Background Aneurysm shrinkage has been proposed as a marker of successful endovascular aneurysm repair (EVAR). Patients with early postoperative shrinkage may experience fewer subsequent complications, and consequently require less intensive surveillance. Methods Patients undergoing EVAR from 2000 to 2011 at three vascular centres (in 2 countries), who had two imaging examinations (postoperative and after 6–18 months), were included. Maximum diameter, complications and secondary interventions during follow-up were registered. Patients were categorized according to early sac dynamics. The primary endpoint was freedom from late complications. Secondary endpoints were freedom from secondary intervention, postimplant rupture and direct (type I/III) endoleaks. Results Some 597 EVARs (71·1 per cent of all EVARs) were included. No shrinkage was observed in 284 patients (47·6 per cent), moderate shrinkage (5–9 mm) in 142 (23·8 per cent) and major shrinkage (at least 10 mm) in 171 patients (28·6 per cent). Four years after the index imaging, the rate of freedom from complications was 84·3 (95 per cent confidence interval 78·7 to 89·8), 88·1 (80·6 to 95·5) and 94·4 (90·1 to 98·7) per cent respectively. No shrinkage was an independent risk factor for late complications compared with major shrinkage (hazard ratio (HR) 3·11; P < 0·001). Moderate compared with major shrinkage (HR 2·10; P = 0·022), early postoperative complications (HR 3·34; P < 0·001) and increasing abdominal aortic aneurysm baseline diameter (HR 1·02; P = 0·001) were also risk factors for late complications. Freedom from secondary interventions and direct endoleaks was greater for patients with major sac shrinkage. Conclusion Early change in aneurysm sac diameter is a strong predictor of late complications after EVAR. Patients with major sac shrinkage have a very low risk of complications for up to 5 years. This parameter may be used to tailor postoperative surveillance. PMID:24752772

  19. Use of inverted intestinal sacs to assess the effect of gastrointestinal insult on carcinogen absorption.

    PubMed

    Capel, I D; Cosier, R S; Pinnock, M H; Williams, D C

    1981-01-01

    Rats were subjected to various forms of treatment in the manner likely to induce gastrointestinal insult. These and control animals were sacrificed and, using inverted sacs, the rate of absorption of either dimethylnitrosamine and benzo(a)pyrene determined. The gastrointestinal injury resulting from the differing treatments did not significantly affect the absorption of benzo(a)pyrene, whereas that of dimethylnitrosamine was significantly increased after each incubation time, most notably by alcohol pretreatment. The results demonstrate that intestinal damage increases the absorption of some carcinogens.

  20. Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists.

    PubMed

    Lockerbie, R O; Hervé, D; Blanc, G; Tassin, J P; Glowinski, J

    1988-01-01

    Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10(-4) M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10(-4) M), vasoactive intestinal polypeptide (10(-6) M) and isoproterenol (10(-5) M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopamine-sensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  2. Macroscopic, cytological and bacteriological evaluation of anal sac content in normal dogs and in dogs with selected dermatological diseases.

    PubMed

    Pappalardo, Ersilia; Martino, Piera Anna; Noli, Chiara

    2002-12-01

    Macroscopic and cytological aspects of anal sac content were evaluated in 40 normal dogs and 10 dogs each with pyoderma, Malassezia dermatitis associated with atopic dermatitis and uncomplicated atopic dermatitis. Bacteria isolated from anal sacs were compared with those from abdominal skin and hair in 20 normal dogs and 10 dogs with pyoderma. There was no difference between the groups in anal sac dimension, or in the colour, consistency or presence of granules in their content. Extracellular bacteria were found in higher numbers in diseased animals, whereas intracellular bacteria were observed in 40% of dogs with pyoderma and in only 2.5% of normal dogs. Malassezia spp. were present in 15.7% of dogs, with no difference between groups. Neutrophils were observed in 12.5% of normal dogs, 30% of dogs with Malassezia dermatitis with underlying atopic dermatitis and in 70 and 80% of dogs with pyoderma and uncomplicated atopic dermatitis, respectively. Seven bacterial species were isolated from anal sacs, with no difference between normal dogs and dogs with pyoderma. In five normal animals and in four dogs with pyoderma the same bacterial strains were isolated from anal sacs and from abdominal skin and hair. PMID:12464064

  3. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions and unsuspected actions.

    PubMed Central

    Giannella, Ralph A.; Mann, Elizabeth A.

    2003-01-01

    Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C. PMID:12813912

  4. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilo