Dysfunction of outer segment guanylate cyclase caused by retinal disease related mutations

PubMed Central

Zägel, Patrick; Koch, Karl-Wilhelm

2014-01-01

Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca2+-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca2+-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca2+-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases. PMID:24616660

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

Glucose and cyclic adenosine monophosphate stimulate activities of adenylate cyclase and guanylate cyclase of Tetrahymena pyriformis infusoria.

PubMed

Shpakov, A O; Derkach, K V; Uspenskaya, Z I

2012-02-01

The sensitivities of cyclase enzymes adenylate cyclase and guanylate cyclase to glucose and extracellular cAMP were studied in Tetrahymena pyriformis infusoria. Glucose effectively stimulated activities of both cyclase enzymes, while cAMP more effectively stimulated adenylate cyclase. It was shown that [6-(14)C]glucose specifically bound to Tetrahymena pyriformis infusoria at dissociation constant (K(D)) and number of binding sites (B(max)) 43 nM and 7.53 fmol glucose per 100,000 cells and [8-(3)H]cAMP bound at 19 nM and 4.46 fmol cAMP per 100,000 cells, respectively. Hence, glucose and cAMP specifically bound to Tetrahymena pyriformis cells and stimulated activities of cyclases in these infusoria.

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

2011-01-04

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).

Soluble guanylate cyclase generation of cGMP regulates migration of MGE neurons.

PubMed

Mandal, Shyamali; Stanco, Amelia; Buys, Emmanuel S; Enikolopov, Grigori; Rubenstein, John L R

2013-10-23

Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2(-/-) and Lhx6(-/-) mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the α subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2(-/-) mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3(-/-) mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity.

BAY 41-2272, a soluble guanylate cyclase stimulator, relaxes isolated human ureter in a standardized in vitro model.

PubMed

Miyaoka, Ricardo; Mendes, Camila; Schenka, André; Gonzalez, Paulo Gabriel; de Nucci, Gilberto; Antunes, Edson; Monga, Manoj; Levi D'Ancona, Carlos Arturo; Mónica, Fabíola Zakia

2014-01-01

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n = 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P <.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved. Copyright © 2014 Elsevier Inc. All rights reserved.

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells.

PubMed

Ali, N; Yousufzai, S Y; Abdel-Latif, A A

2000-07-01

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

PubMed

Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue.

PubMed

Gray, Jesse M; Karow, David S; Lu, Hang; Chang, Andy J; Chang, Jennifer S; Ellis, Ronald E; Marletta, Michael A; Bargmann, Cornelia I

2004-07-15

Specialized oxygen-sensing cells in the nervous system generate rapid behavioural responses to oxygen. We show here that the nematode Caenorhabditis elegans exhibits a strong behavioural preference for 5-12% oxygen, avoiding higher and lower oxygen levels. 3',5'-cyclic guanosine monophosphate (cGMP) is a common second messenger in sensory transduction and is implicated in oxygen sensation. Avoidance of high oxygen levels by C. elegans requires the sensory cGMP-gated channel tax-2/tax-4 and a specific soluble guanylate cyclase homologue, gcy-35. The GCY-35 haem domain binds molecular oxygen, unlike the haem domains of classical nitric-oxide-regulated guanylate cyclases. GCY-35 and TAX-4 mediate oxygen sensation in four sensory neurons that control a naturally polymorphic social feeding behaviour in C. elegans. Social feeding and related behaviours occur only when oxygen exceeds C. elegans' preferred level, and require gcy-35 activity. Our results suggest that GCY-35 is regulated by molecular oxygen, and that social feeding can be a behavioural strategy for responding to hyperoxic environments.

The effects of the soluble guanylate cyclase stimulator riociguat on memory performance in healthy volunteers with a biperiden-induced memory impairment.

PubMed

Borghans, Laura G J M; Sambeth, Anke; Prickaerts, Jos; Ramaekers, Johannes G; Blokland, Arjan

2018-06-07

After stimulation with nitric oxide, soluble guanylate cyclase (sGC) produces cyclic guanosine monophosphate (cGMP), which stimulates an important signalling pathway for long-term potentiation (LTP). By upregulating cGMP, LTP could be stimulated and thereby enhancing memory processes. The present study investigated the effects of the sGC stimulator riociguat on cognition in healthy volunteers. Participants were pre-treated with and without biperiden, which impairs memory performance, to investigate the memory-enhancing effects of riociguat. Twenty volunteers participated in a double-blind placebo-controlled six-way crossover design with a cognitive test battery including the verbal learning task (VLT), n-back task, spatial memory test, the attention network test, and a reaction time task. Treatments were placebo and riociguat 0.5 mg, placebo and riociguat 1.0 mg, biperiden 2.0 mg and placebo, biperiden 2.0 mg and riociguat 0.5 mg and biperiden 2.0 mg and riociguat 1.0 mg. Blood pressure was found to be decreased and heart rate to be increased after administration of riociguat. Cognitive performance was not enhanced after administration of riociguat. Biperiden decreased episodic memory on the VLT, yet this deficit was not reversed by riociguat. This supports the notion that biperiden might be a valuable pharmacological model to induce episodic memory impairments as observed in AD/MCI.

High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation.

PubMed

Jebelovszki, Eva; Kiraly, Csaba; Erdei, Nora; Feher, Attila; Pasztor, Eniko T; Rutkai, Ibolya; Forster, Tamas; Edes, Istvan; Koller, Akos; Bagi, Zsolt

2008-06-01

The impact of obesity on nitric oxide (NO)-mediated coronary microvascular responses is poorly understood. Thus NO-mediated vasomotor responses were investigated in pressurized coronary arterioles ( approximately 100 microm) isolated from lean (on normal diet) and obese (fed with 60% of saturated fat) rats. We found that dilations to acetylcholine (ACh) were not significantly different in obese and lean rats (lean, 83 +/- 4%; and obese, 85 +/- 3% at 1 microM), yet the inhibition of NO synthesis with N(omega)-nitro-l-arginine methyl ester reduced ACh-induced dilations only in vessels of lean controls. The presence of the soluble guanylate cyclase (sGC) inhibitor oxadiazolo-quinoxaline (ODQ) elicited a similar reduction in ACh-induced dilations in the two groups of vessels (lean, 60 +/- 11%; and obese, 57 +/- 3%). Dilations to NO donors, sodium nitroprusside (SNP), and diethylenetriamine (DETA)-NONOate were enhanced in coronary arterioles of obese compared with lean control rats (lean, 63 +/- 6% and 51 +/- 5%; and obese, 78 +/- 5% and 70 +/- 5%, respectively, at 1 microM), whereas dilations to 8-bromo-cGMP were not different in the two groups. In the presence of ODQ, both SNP and DETA-NONOate-induced dilations were reduced to a similar level in lean and obese rats. Moreover, SNP-stimulated cGMP immunoreactivity in coronary arterioles and also cGMP levels in carotid arteries were enhanced in obese rats, whereas the protein expression of endothelial NOS and the sGC beta1-subunit were not different in the two groups. Collectively, these findings suggest that in coronary arterioles of obese rats, the increased activity of sGC leads to an enhanced sensitivity to NO, which may contribute to the maintenance of NO-mediated dilations and coronary perfusion in obesity.

Soluble guanylate cyclase activation during ischemic injury in mice protects against postischemic inflammation at the mitochondrial level.

PubMed

Wang, Derek Z; Jones, Allan W; Wang, Walter Z; Wang, Meifang; Korthuis, Ronald J

2016-05-01

The aim was to determine whether treatment with BAY 60-2770, a selective activator of oxidized soluble guanylate cyclase (sGC), near the end of an ischemic event would prevent postischemic inflammation and mitochondrial dysfunction in wild-type (WT) and heme oxygenase-1 KO (HO-1(-/-)) mice. This protocol prevented increases in leukocyte rolling (LR) and adhesion (LA) to intestinal venules along with elevated TNFα and circulating neutrophil levels that accompany ischemia-reperfusion (I/R) in both animal models. We further hypothesized that a component of BAY 60-2770 treatment involves maintenance of mitochondrial membrane integrity during I/R. Measurements on isolated enterocytes of calcein fluorescence (mitochondrial permeability) and JC-1 fluorescence ratio (mitochondrial membrane potential) were reduced by I/R, indicating formation of mitochondrial permeability transition pores (mPTP). These effects were abrogated by BAY 60-2770 as well as cyclosporin A and SB-216763, which prevented mPTP opening and inhibited glycogen synthase kinase-3β (GSK-3β), respectively. Western blots of WT and HO-1(-/-) enterocytes indicated that GSK-3β phosphorylation on Ser(9) (inhibitory site) was reduced by half following I/R alone (increased GSK-3β activity) and increased by one-third (reduced GSK-3β activity) following BAY 60-2770. Other investigators have associated phosphorylation of the GSK-3β substrate cyclophilin D (pCyPD) with mPTP formation. We observed a 60% increase in pCyPD after I/R, whereas BAY 60-2770 treatment of sham and I/R groups reduced pCyPD by about 20%. In conclusion, selective activation of oxidized sGC of WT and HO-1(-/-) during ischemia protects against I/R-induced inflammation and preserves mucosal integrity in part by reducing pCyPD production and mPTP formation. Copyright © 2016 the American Physiological Society.

Oxygen Binding and Redox Properties of the Heme in Soluble Guanylate Cyclase

PubMed Central

Makino, Ryu; Park, Sam-yon; Obayashi, Eiji; Iizuka, Tetsutaro; Hori, Hiroshi; Shiro, Yoshitugu

2011-01-01

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O2, thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O2 has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe2+-O2) when frozen at 77 K in the presence of O2. The ligation of O2 was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at −7 °C, with the extremely low affinity for O2. The low O2 affinity was not caused by a distal steric protein effect and by rupture of the Fe2+-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe2+-proximal His bond. Based on our results, we propose that the weak Fe2+-proximal His bond is a key determinant for the low O2 affinity of the heme moiety of soluble guanylate cyclase. PMID:21385878

N-hydroxylamine is not an intermediate in the conversion of L-arginine to an activator of soluble guanylate cyclase in neuroblastoma N1E-115 cells.

PubMed Central

Pou, S; Pou, W S; Rosen, G M; el-Fakahany, E E

1991-01-01

This study evaluates the role of N-hydroxylamine (NH2OH) in activating soluble guanylate cyclase in the mouse neuroblastoma clone N1E-115. It has been proposed that NH2OH is a putative intermediate in the biochemical pathway for the generation of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) from L-arginine. NH2OH caused a time- and concentration-dependent increase in cyclic GMP formation in intact cells. This response was not dependent on Ca2+. In cytosol preparations the activation of guanylate cyclase by L-arginine was dose-dependent and required Ca2+ and NADPH. In contrast, NH2OH itself did not activate cytosolic guanylate cyclase but it inhibited the basal activity of this enzyme in a concentration-dependent manner. The formation of cyclic GMP in the cytosolic fractions in response to NH2OH required the addition of catalase and H2O2. On the other hand, catalase and/or H2O2 lead to a decrease in L-arginine-induced cyclic GMP formation. Furthermore, NH2OH inhibited L-arginine- and sodium nitroprusside-induced cyclic GMP formation in the cytosol. The inhibition of L-arginine-induced cyclic GMP formation in the cytosol by NH2OH was not reversed by the addition of superoxide dismutase. These data strongly suggest that NH2OH is not a putative intermediate in the metabolism of L-arginine to an activator of guanylate cyclase. PMID:1671745

Human trabecular meshwork cell volume decrease by NO-independent soluble guanylate cyclase activators YC-1 and BAY-58-2667 involves the BKCa ion channel.

PubMed

Dismuke, William M; Sharif, Najam A; Ellis, Dorette Z

2009-07-01

There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3',5'-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BK(Ca) channel were used to characterize their involvement in the YC-1- and BAY-58-2667-induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay. YC-1 (10 nM-200 microM) and BAY-58-2667 (10 nM-100 microM) each elicited a biphasic effect on TM cell volume. YC-1 (1 microM) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1-induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BK(Ca) channel inhibitor IBTX. The BAY-58-2667-induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BK(Ca) channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667-induced decreases in cell volume. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K(+) efflux.

Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina.

PubMed

López-Begines, Santiago; Plana-Bonamaisó, Anna; Méndez, Ana

2018-02-13

Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekiya, M.; Frohlich, E.D.; Cole, F.E.

1991-01-01

In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation.more » In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.« less

Stabilizing function for myristoyl group revealed by the crystal structure of a neuronal calcium sensor, guanylate cyclase-activating protein 1.

PubMed

Stephen, Ricardo; Bereta, Grzegorz; Golczak, Marcin; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2007-11-01

Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins myristoylated at the N terminus that regulate guanylate cyclases in photoreceptor cells and belong to the family of neuronal calcium sensors (NCS). Many NCS proteins display a recoverin-like "calcium-myristoyl switch" whereby the myristoyl group, buried inside the protein in the Ca(2+)-free state, becomes fully exposed upon Ca(2+) binding. Here we present a 2.0 A resolution crystal structure of myristoylated GCAP1 with Ca(2+) bound. The acyl group is buried inside Ca(2+)-bound GCAP1. This is in sharp contrast to Ca(2+)-bound recoverin, where the myristoyl group is solvent exposed. Furthermore, we provide direct evidence that the acyl group in GCAP1 remains buried in the Ca(2+)-free state and does not undergo switching. A pronounced kink in the C-terminal helix and the presence of the myristoyl group allow clustering of sequence elements crucial for GCAP1 activity.

Stabilizing Function for Myristoyl Group Revealed by the Crystal Structure of a Neuronal Calcium Sensor, Guanylate Cyclase-Activating Protein 1

PubMed Central

Stephen, Ricardo; Bereta, Grzegorz; Golczak, Marcin; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2008-01-01

SUMMARY Guanylate cyclase-activating proteins (GCAPs) are Ca2+-binding proteins myristoylated at the N terminus that regulate guanylate cyclases in photoreceptor cells and belong to the family of neuronal calcium sensors (NCS). Many NCS proteins display a recoverin-like “calcium-myristoyl switch” whereby the myristoyl group, buried inside the protein in the Ca2+-free state, becomes fully exposed upon Ca2+ binding. Here we present a 2.0 Å resolution crystal structure of myristoylated GCAP1 with Ca2+ bound. The acyl group is buried inside Ca2+-bound GCAP1. This is in sharp contrast to Ca2+-bound recoverin, where the myristoyl group is solvent exposed. Furthermore, we provide direct evidence that the acyl group in GCAP1 remains buried in the Ca2+-free state and does not undergo switching. A pronounced kink in the C-terminal helix and the presence of the myristoyl group allow clustering of sequence elements crucial for GCAP1 activity. PMID:17997965

Nitric oxide in B6 mouse and nitric oxide-sensitive soluble guanylate cyclase in cat modulate acetylcholine release in pontine reticular formation.

PubMed

Lydic, Ralph; Garza-Grande, Ricardo; Struthers, Richard; Baghdoyan, Helen A

2006-05-01

ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly (P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release (P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant (P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Direct sGC Activation Bypasses NO Scavenging Reactions of Intravascular Free Oxy-Hemoglobin and Limits Vasoconstriction

PubMed Central

Tabima, D. Marcela; Specht, Patricia A.C.; Tejero, Jesús; Champion, Hunter C.; Kim-Shapiro, Daniel B.; Baust, Jeff; Mik, Egbert G.; Hildesheim, Mariana; Stasch, Johannes-Peter; Becker, Eva-Maria; Truebel, Hubert

2013-01-01

Abstract Aims: Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. Results: Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. Innovation and Conclusion: These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling. Antioxid. Redox Signal. 19, 2232–2243. PMID:23697678

The Impact of the Nitric Oxide (NO)/Soluble Guanylyl Cyclase (sGC) Signaling Cascade on Kidney Health and Disease: A Preclinical Perspective.

PubMed

Krishnan, Shalini M; Kraehling, Jan R; Eitner, Frank; Bénardeau, Agnès; Sandner, Peter

2018-06-09

Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators.

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

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

PubMed

Vinberg, Frans; Peshenko, Igor V; Chen, Jeannie; Dizhoor, Alexander M; Kefalov, Vladimir J

2018-05-11

Light adaptation of photoreceptor cells is mediated by Ca 2+ -dependent mechanisms. In darkness, Ca 2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca 2+ extrusion via Na + /Ca 2+ , K + exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca 2+ levels in photoreceptor outer segment because of continuing Ca 2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca 2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca 2+ -feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca 2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca 2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

PubMed

Etzl, Stefan; Lindner, Robert; Nelson, Matthew D; Winkler, Andreas

2018-06-08

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties. © 2018 Etzl et al.

Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity*

PubMed Central

Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K.; Makino, Clint L.

2015-01-01

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 [Ca2+]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 Ca2+ nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca2+]. 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

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.

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Toll-like Receptor 4 Signaling Confers Cardiac Protection Against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms

PubMed Central

Wang, E; Feng, Yan; Zhang, Ming; Zou, Lin; Li, Yan; Buys, Emmanuel S.; Huang, Peigen; Brouckaert, Peter; Chao, Wei

2011-01-01

Background Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that TLR4 mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon-β–mediated transcription-factor (Trif), inducible nitric-oxide synthase (iNOS), and soluble guanylate cyclase (sGC). Methods Wild-type mice and the genetically modified mice, i.e., TLR4-deficient (TLR4-def), TLR2 knockout (TLR2−/−), MyD88−/−, Trif−/−, iNOS−/−, and sGCα1−/−, were treated with normal saline or 0.1 mg/kg of lipopolysaccharide, intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min of global ischemia and reperfusion for up to 60 min. Left ventricular function and myocardial infarction sizes were examined. Results Compared to saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dtmax (P < 0.01) and reduced MI sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4-def and MyD88−/− mice, but remained intact in TLR2−/− or Trif−/− mice. iNOS−/− mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. While sGC 1−/− mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection. Conclusions TLR4 activation confers a potent cardiac protection against ischemia

Membrane guanylate cyclase, a multimodal transduction machine: history, present, and future directions

PubMed Central

Sharma, Rameshwar K.; Duda, Teresa

2014-01-01

A sequel to these authors' earlier comprehensive reviews which covered the field of mammalian membrane guanylate cyclase (MGC) from its origin to the year 2010, this article contains 13 sections. The first is historical and covers MGC from the year 1963–1987, summarizing its colorful developmental stages from its passionate pursuit to its consolidation. The second deals with the establishment of its biochemical identity. MGC becomes the transducer of a hormonal signal and founder of the peptide hormone receptor family, and creates the notion that hormone signal transduction is its sole physiological function. The third defines its expansion. The discovery of ROS-GC subfamily is made and it links ROS-GC with the physiology of phototransduction. Sections ROS-GC, a Ca2+-Modulated Two Component Transduction System to Migration Patterns and Translations of the GCAP Signals Into Production of Cyclic GMP are Different cover its biochemistry and physiology. The noteworthy events are that augmented by GCAPs, ROS-GC proves to be a transducer of the free Ca2+ signals generated within neurons; ROS-GC becomes a two-component transduction system and establishes itself as a source of cyclic GMP, the second messenger of phototransduction. Section ROS-GC1 Gene Linked Retinal Dystrophies demonstrates how this knowledge begins to be translated into the diagnosis and providing the molecular definition of retinal dystrophies. Section Controlled By Low and High Levels of [Ca2+]i, ROS-GC1 is a Bimodal Transduction Switch discusses a striking property of ROS-GC where it becomes a “[Ca2+]i bimodal switch” and transcends its signaling role in other neural processes. In this course, discovery of the first CD-GCAP (Ca2+-dependent guanylate cyclase activator), the S100B protein, is made. It extends the role of the ROS-GC transduction system beyond the phototransduction to the signaling processes in the synapse region between photoreceptor and cone ON-bipolar cells; in section Ca2

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

PubMed

Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

2010-07-01

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX.

PubMed

elDeib, M M; Parker, C D; White, A A

1987-04-02

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins.

PubMed

Pattison, Amanda M; Blomain, Erik S; Merlino, Dante J; Wang, Fang; Crissey, Mary Ann S; Kraft, Crystal L; Rappaport, Jeff A; Snook, Adam E; Lynch, John P; Waldman, Scott A

2016-10-01

Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Activation of intestinal brush border guanylate cyclase by aromatic disulphide compounds.

PubMed Central

elDeib, M M; Parker, C D; White, A A

1991-01-01

Guanylate cyclase in pig intestinal brush border membranes was stimulated by certain aromatic disulphides. The most effective were 6-thioguanine disulphide [(TGS)2], 6-mercaptopurine disulphide, 6,6'-dithiodinicotinic acid, 5,5'-dithiobis-(2-nitrobenzoic acid) and 5-carboxy-2-thiouracil disulphide. (TGS)2 stimulated activity 15-fold when present at 0.1 mM. The optimum concentration for each disulphide was different, and higher concentrations were inhibitory. There was no activation by alkyl disulphides or by N-ethylmaleimide. Activation by 50 microM-(TGS)2 was partially reversed by later addition of 0.1 mM-dithiothreitol, whereas activation by the Escherichia coli heat-stable enterotoxin STa was relatively unaffected. Pretreatment of the membranes with (TGS)2 produced a concentration-dependent inhibition of STa-stimulated activity, while stimulating basal activity, until the activities were equal at 50 microM. Activity was [Mg2+]-dependent, the optimal [Mg2+] progressively increasing as the enzyme was stimulated by (TGS)2, STa and Lubrol PX respectively. However, (TGS)2 pretreatment prevented the shift to higher [Mg2+]optima induced by STa or Lubrol alone. Substitution of Mn2+ for Mg2+ in the reaction elevated basal activity and eliminated by activation (TGS)2. (TGS)2 only inhibited Mn2(+)-dependent activity (both basal and stimulated). The affinity of 125I-STa for its receptor was slightly increased by (TGS)2. We propose that (TGS)2 undergoes thiol-disulphide exchange with at least three different protein thiols of decreasing reactivity. The first is associated with Mg2(+)-dependent activation, the second is associated with a tonic inhibition of activity and the third is associated with the catalytic activity, although probably not at the active site. PMID:1673335

ATRIAL NATRIURETIC FACTOR RECEPTOR GUANYLATE CYCLASE SIGNALING: NEW ATP- REGULATED TRANSDUCTION MOTIF

PubMed Central

Duda, Teresa; Bharill, Shashank; Wojtas, Ireneusz; Yadav, Prem; Gryczynski, Ignacy; Gryczynski, Zygmunt; Sharma, Rameshwar K.

2010-01-01

ANF-RGC$ membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques ofsteady-state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains themechanism of the model-based predicted transduction role of the ARM’s structural motif, 669WTAPELL675. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the 669WTAPELL675 motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC. PMID:19137266

Vasodilator effects and putative guanylyl cyclase stimulation by 2-nitro-1-phenylethanone and 2-nitro-2-phenyl-propane-1,3-diol on rat aorta.

PubMed

Vasconcelos, Thiago Brasileiro de; Ribeiro-Filho, Helder Veras; Lahlou, Saad; Pereira, José Geraldo de Carvalho; Oliveira, Paulo Sérgio Lopes de; Magalhães, Pedro Jorge Caldas

2018-07-05

Compounds containing a nitro group may reveal vasodilator properties. Several nitro compounds have a NO 2 group in a short aliphatic chain connected to an aromatic group. In this study, we evaluated in rat aorta the effects of two nitro compounds, with emphasis on a putative recruitment of the soluble guanylate cyclase (sGC) pathway to induce vasodilation. Isolated aortic rings were obtained from male Wistar rats to compare the effects induced by 2-nitro-1-phenylethanone (NPeth) or 2-nitro-2-phenyl-propane-1,3-diol (NPprop). In aortic preparations contracted with phenylephrine or KCl, NPeth and NPprop induced vasorelaxant effects that did not depend on the integrity of vascular endothelium. NPeth had a lesser vasorelaxant efficacy than NPprop and only the NPprop effects were inhibited by pretreatment with the sGC inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue. In an ODQ-preventable manner, NPprop inhibited the contractile component of the phenylephrine-induced response mediated by intracellular Ca 2+ release or by extracellular Ca 2+ recruitment through receptor- or voltage-operated Ca 2+ channels. In contrast, NPprop was inert against the transient contraction induced by caffeine in Ca 2+ -free medium. In an ODQ-dependent manner, NPprop inhibited the contraction induced by the protein kinase C activator phorbol 12,13-dibutyrate or by the tyrosine phosphatase inhibitor sodium orthovanadate. In silico docking analysis of a sGC homologous protein revealed preferential site for NPprop. In conclusion, the nitro compounds NPeth and NPprop induced vasorelaxation in rat aortic rings. Aliphatic chain substituents selectively interfered in the ability of these compounds to induce vasorelaxant effects, and only NPprop relaxed aortic rings via a sGC pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric oxide synthase and soluble guanylate cyclase are involved in spinal cord wind-up activity of monoarthritic, but not of normal rats.

PubMed

Laurido, Claudio; Hernández, Alejandro; Constandil, Luis; Pelissier, Teresa

2003-11-27

While increasing evidence points to a role for the nitric oxide (NO)/cyclic guanosine 3,5-monophosphate (GMPc) cascade in hyperalgesia and allodynia, participation of the NO/GMPc pathway in synaptic processing in the spinal cord, i.e. wind-up activity, is less clear. We studied the effects of intrathecal administration of Nomega-nitro-L-arginine methyl ester (L-NAME) and methylene blue, inhibitors of NO synthase and guanylate cyclase respectively, on wind-up activity developed in a C-fiber reflex response paradigm. 5, 10 and 20 microg i.t. of L-NAME or methylene blue did not modify spinal wind-up in normal rats, while a dose-dependent inhibition of wind-up was observed in monoarthritic rats. Results suggest that the NO/GMPc pathway plays a non-significant role in wind-up activity evoked in normal animals, while it may be essential in chronic pain processing.

Targeting transcriptional control of soluble guanylyl cyclase via NOTCH for prevention of cardiovascular disease.

PubMed

Rippe, Catarina; Albinsson, Sebastian; Guron, Gregor; Nilsson, Holger; Swärd, Karl

2018-05-13

Soluble guanylyl cyclase (sGC) is an effector enzyme of nitric oxide (NO). Recent work has unraveled how levels of this enzyme are controlled, and highlighted a role in vascular disease. We provide a timely summary of available knowledge on transcriptional regulation of sGC, including influences from the NOTCH signaling pathway and genetic variants. It is speculated that hypertension-induced repression of sGC starts a vicious circle that can be initiated by periods of stress, diet or genetic factors, and a key tenet is that reduction of sGC further raises blood pressure. The idea that dysregulation of sGC contributes to syndromes caused by defective NOTCH signaling is advanced, and we discuss drug repositioning for vascular disease prevention. The advantage of targeting sGC expression rather than activity is also considered. It is argued that transcriptional inputs on sGC arise from interactions with other cells, the extracellular matrix, and microRNAs (miRNAs), and concluded that the promise of sGC as a target for prevention of cardiovascular disease has increased in recent time. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misono, K. S.; Philo, J. S.; Arakawa, T.

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 ofmore » 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.« less

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies

PubMed Central

Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

2015-01-01

Background and Purpose Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. Experimental Approach High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Key Results Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. Conclusions and Implications These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. PMID:25536881

The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies.

PubMed

Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

2015-05-01

Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. © 2014 The British Pharmacological Society.

Thrombospondin-1 and Angiotensin II Inhibit Soluble Guanylyl Cyclase through an Increase in Intracellular Calcium Concentration

PubMed Central

Ramanathan, Saumya; Mazzalupo, Stacy; Boitano, Scott; Montfort, William R.

2011-01-01

Nitric Oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca2+]i) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca2+]i. Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca2+]i, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca2+]i, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca2+ remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in Km for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca2+]i in response to NO, inducing vasodilation, but is also inhibited by high [Ca2+]i, providing a fine balance between signals for vasodilation and vasoconstriction. PMID:21823650

Role of sulfhydryl-dependent dimerization of soluble guanylyl cyclase in relaxation of porcine coronary artery to nitric oxide.

PubMed

Zheng, Xiaoxu; Ying, Lei; Liu, Juan; Dou, Dou; He, Qiong; Leung, Susan Wai Sum; Man, Ricky Y K; Vanhoutte, Paul M; Gao, Yuansheng

2011-06-01

Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO). The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine. These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.

Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats.

PubMed

Jabs, A; Oelze, M; Mikhed, Y; Stamm, P; Kröller-Schön, S; Welschof, P; Jansen, T; Hausding, M; Kopp, M; Steven, S; Schulz, E; Stasch, J-P; Münzel, T; Daiber, A

2015-08-01

Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100mg/kg/d for 3.5days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5mg/kg/d) or BAY 41-8543 (1 and 5mg/kg/d) for 6days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3-nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance. Copyright © 2015 Elsevier Inc. All rights reserved.

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

PubMed

Glynos, Constantinos; Toumpanakis, Dimitris; Loverdos, Konstantinos; Karavana, Vassiliki; Zhou, Zongmin; Magkou, Christina; Dettoraki, Maria; Perlikos, Fotis; Pavlidou, Athanasia; Kotsikoris, Vasilis; Topouzis, Stavros; Theocharis, Stamatios E; Brouckaert, Peter; Giannis, Athanassios; Papapetropoulos, Andreas; Vassilakopoulos, Theodoros

2015-06-01

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

PubMed

Chen, Zhengju; Zhang, Xu; Ying, Lei; Dou, Dou; Li, Yanhui; Bai, Yun; Liu, Juan; Liu, Limei; Feng, Han; Yu, Xiaoxing; Leung, Susan Wai-Sum; Vanhoutte, Paul M; Gao, Yuansheng

2014-08-01

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK. Copyright © 2014 the American Physiological Society.

Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.

PubMed

Shailubhai, Kunwar; Palejwala, Vaseem; Arjunan, Krishna Priya; Saykhedkar, Sayali; Nefsky, Bradley; Foss, John A; Comiskey, Stephen; Jacob, Gary S; Plevy, Scott E

2015-11-06

To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα(-/-)) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα(-/-) mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. This is the first-ever study reporting

Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis

PubMed Central

Shailubhai, Kunwar; Palejwala, Vaseem; Arjunan, Krishna Priya; Saykhedkar, Sayali; Nefsky, Bradley; Foss, John A; Comiskey, Stephen; Jacob, Gary S; Plevy, Scott E

2015-01-01

AIM: To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. METHODS: The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα-/-) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα-/- mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is

CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

NASA Technical Reports Server (NTRS)

Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

2003-01-01

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

PubMed

Cabilla, Jimena P; Díaz, María del Carmen; Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2006-09-01

Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

Nitric oxide contributes to high-salt perception in a blood-sucking insect model.

PubMed

Cano, Agustina; Pontes, Gina; Sfara, Valeria; Anfossi, Diego; Barrozo, Romina B

2017-11-14

In all organisms, salts produce either appetitive or aversive responses depending on the concentration. While low-salt concentration in food elicits positive responses to ingest, high-salt triggers aversion. Still the mechanisms involved in this dual behavior have just started to be uncovered in some organisms. In Rhodnius prolixus, using pharmacological and behavioral assays, we demonstrated that upon high-salt detection in food a nitric oxide (NO) dependent cascade is activated. This activation involves a soluble guanylate cyclase (sGC) and the production of cyclic guanosine monophosphate (cGMP). Thus, appetitive responses to low-salt diets turn to aversion whenever this cascade is activated. Conversely, insects feed over aversive high-salt solutions when it is blocked by reducing NO levels or by affecting the sGC activity. The activation of NO/sGC/cGMP cascade commands the avoidance feeding behavior in R. prolixus. Investigations in other insect species should examine the possibility that high-salt aversion is mediated by NO/sSG/cGMP signaling.

Preclinical Evaluation of a Replication-Deficient Recombinant Adenovirus Serotype 5 Vaccine Expressing Guanylate Cyclase C and the PADRE T-helper Epitope

PubMed Central

Snook, Adam E.; Baybutt, Trevor R.; Hyslop, Terry; Waldman, Scott A.

2016-01-01

There is an unmet need for improved therapeutics for colorectal cancer, the second leading cause of cancer mortality worldwide. Adjuvant chemotherapy only marginally improves survival in some patients and has no benefit in others, underscoring the clinical opportunity for novel immunotherapeutic approaches to improve survival in colorectal cancer. In that context, guanylate cyclase C (GUCY2C) is an established biomarker and therapeutic target for metastatic colorectal cancer with immunological characteristics that promote durable antitumor efficacy without autoimmunity. Preliminary studies established non-replicating human type 5 adenovirus (Ad5) expressing GUCY2C as safe and effective to induce GUCY2C-specific immune responses and antitumor immunity in mice. This study characterized the biodistribution, immunogenicity, and safety of a vector expressing GUCY2C fused with the human CD4+ T helper cell epitope PADRE (Ad5-GUCY2C-PADRE) to advance this vaccine into clinical trials in colorectal cancer patients. Ad5-GUCY2C-PADRE levels were highest in the injection site and distributed in vivo primarily to draining lymph nodes, the liver, spleen and, unexpectedly, to the bone marrow. Immune responses following Ad5-GUCY2C-PADRE administration were characterized by PADRE-specific CD4+ T-cell and GUCY2C-specific B-cell and CD8+ T-cell responses, producing antitumor immunity targeting GUCY2C-expressing colorectal cancer metastases in the lungs, without acute or chronic autoimmune or other toxicities. Collectively, these data support Ad5-GUCY2C-PADRE as a safe and effective vaccination strategy in preclinical models and position Ad5-GUCY2C-PADRE for Phase I clinical testing in colorectal cancer patients. PMID:27903079

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

PubMed

Lee, Nikki P Y; Cheng, C Yan

2003-07-01

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ

Apoptosis of human gastric cancer SGC-7901 cells induced by podophyllotoxin.

PubMed

Ji, Chen-Feng; Ji, Yu-Bin

2014-05-01

Numerous studies have demonstrated that podophyllotoxin and its derivatives exhibit antitumor effects. The aim of the present study was to investigate SGC-7901 cell apoptosis and the underlying mechanism induced by podophyllotoxin. SGC-7901 cells were treated with varying concentrations of podophyllotoxin. MTT assays and flow cytometry were used to evaluate the effects of podophyllotoxin on the proliferation and apoptosis of SGC-7901 cells, while fluorescence inverted microscopy was used to observe the morphology of SGC-7901 cells that had been dyed with Hoechst 33258. In addition, laser scanning confocal microscopy was used to analyze the mitochondrial membrane potential (MMP) of SGC-7901 cells dyed with Rhodamine 123. Western blotting was performed to analyze the expression levels of cytochrome c (cyt- c ), caspase-9 and caspase-3 in the SGC-7901 cells. The results indicated that podophyllotoxin was capable of inhibiting growth and inducing the apoptosis of SGC-7901 cells in a dose-dependent manner, causing cell cycle arrest at the G 2 /M phase. After 48 h of treatment, the apoptotic morphology of SGC-7901 cells was clear, exhibiting cell protuberance, concentrated cytoplasms and apoptotic bodies. Following 24 h of treatment, the MMP of the SGC-7901 cells decreased. In addition, after 48 h, the expression of cyt- c was shown to be upregulated, while the expression levels of pro-caspase-9 and pro-caspase-3 in the SGC-7901 cells were shown to be downregulated. In conclusion, apoptosis can be induced in SGC-7901 cells by podophyllotoxin, potentially via a mitochondrial pathway, indicating that podophyllotoxin may be a potent agent for cancer treatment.

Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone.

PubMed

Detremmerie, Charlotte; Vanhoutte, Paul M; Leung, Susan

2017-07-01

The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine 3',5'-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

PubMed Central

Lakshminrusimha, Satyan; Wedgwood, Stephen; Czech, Lyubov; Gugino, Sylvia F.; Russell, James A.; Farrow, Kathryn N.; Steinhorn, Robin H.

2012-01-01

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O2 for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg−1·dose−1 times two doses (PPHN-loHC), and six lambs were ventilated with O2 alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O2 ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O2. We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress. PMID:22198909

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

PubMed

Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier ProteinTethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus

DOE PAGES

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan; ...

2016-08-25

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

PubMed

Cabilla, Jimena P; Nudler, Silvana I; Ronchetti, Sonia A; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

2011-01-01

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway. © 2011 Cabilla et al.

Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase.

PubMed

Wu, Gang; Liu, Wen; Berka, Vladimir; Tsai, Ah-Lim

2017-09-01

To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O 2 , including K D , k on , and k off , of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The K D (NO) and K D (CO) of each bacterial H-NOX or sGC follow the "sliding scale rule"; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain

PubMed Central

VIT, JEAN-PHILIPPE; JASMIN, LUC; BHARGAVA, ADITI; OHARA, PETER T.

2008-01-01

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K+ channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca2+-activated K+ channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K+) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain. PMID:18568096

Contemporary approaches to modulating the nitric oxide-cGMP pathway in cardiovascular disease

PubMed Central

Kraehling, Jan R.; Sessa, William C.

2017-01-01

Endothelial cells lining the vessel wall control important aspects of vascular homeostasis. In particular, the production of endothelium-derived nitric oxide and activation of soluble guanylate cyclase promotes endothelial quiescence and governs vasomotor function and proportional remodeling of blood vessels. Here, we discuss novel approaches to improve endothelial nitric oxide generation and preserve its bioavailability. We also discuss therapeutic opportunities aimed at activation of soluble guanylate cyclase for multiple cardiovascular indications. PMID:28360348

Preservation of nitric oxide-induced relaxation of porcine coronary artery: roles of the dimers of soluble guanylyl cyclase, phosphodiesterase type 5, and cGMP-dependent protein kinase.

PubMed

Liu, Juan; Chen, Zhengju; Ye, Liping; Liu, Huixia; Dou, Dou; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng

2014-10-01

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3'5'-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.

Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos).

PubMed

Yang, S; Fan, R; Shi, Z; Ji, K; Zhang, J; Wang, H; Herrid, M; Zhang, Q; Yao, J; Smith, G W; Dong, C

2015-04-01

The molecular mechanisms underlying the formation of coat colors in animals are poorly understood. Recent studies have demonstrated that microRNA play important roles in the control of melanogenesis and coat color in mammals. In a previous study, we characterized the miRNA expression profiles in alpaca skin with brown and white coat color and identified a novel miRNA (named lpa-miR-nov-66) that is expressed significantly higher in white skin compared to brown skin. The present study was conducted to determine the functional roles of this novel miRNA in the regulation of melanogenesis in alpaca melanocytes. lpa-miR-nov-66 is predicted to target the soluble guanylate cyclase (sGC) gene based on presence of a binding site in the sGC coding sequence (CDS). Overexpression of lpa-miR-nov-66 in alpaca melanocyes upregulated the expression of sGC both at the mRNA and protein level. Overexpression of lpa-miR-nov-66 in melanocyes also resulted in decreased expression of key melanogenic genes including tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and microphthalmia transcription factor (MITF). Our ELISA assays showed increased cyclic guanosine monophosphate (cGMP) but decreased cyclic adenosine monophosphate (cAMP) production in melanocytes overexpressing lpa-miR-nov-66. In addition, overexpression of lpa-miR-nov-66 also reduced melanin production in cultured melanocytes. Results support a role of lpa-miR-nov-66 in melanocytes by directly or indirectly targeting , which regulates melanogenesis via the cAMP pathway.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

PubMed

Opelt, Marissa; Eroglu, Emrah; Waldeck-Weiermair, Markus; Russwurm, Michael; Koesling, Doris; Malli, Roland; Graier, Wolfgang F; Fassett, John T; Schrammel, Astrid; Mayer, Bernd

2016-11-11

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibitory effect of acetylshikonin on human gastric carcinoma cell line SGC-7901 in vitro and in vivo

PubMed Central

Zeng, Yun; Liu, Gang; Zhou, Li-Ming

2009-01-01

AIM: To investigate the inhibitory effect of acetylshikonin on human gastric carcinoma cell line SGC-7901 and its mechanism. METHODS: MTT assay was used to assess the inhibitory effect of acetylshikonin on proliferation of SGC-7901 cells. Apoptosis-inducing effect was determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling with Hoechst staining. Expression of mRNA and protein in Bcl-2 and Bax was analyzed by reverse transcription-polymerase chain reaction and Western blot. Antitumor effect of acetylshikonin on a mouse SGC-7901 model was also determined. RESULTS: Forty-eight hours after treatment with acetylshikonin, MTT assay showed that acetylshikonin inhibited the proliferation of SGC-7901 cells in a dose-dependent manner. The half maximal inhibitory concentration of acetylshikonin to SGC-7901 cells was 0.428 ± 0.07 mg/L. Cell shrinkage, nuclear pyknosis and chromatin condensation, which are the characteristics of cell apoptosis, were observed in treated SGC-7901 cells and the percentage of apoptosis increased in a dose-dependent manner. Acetylshikonin down-regulated the expression of Bcl-2 and up-regulated the expression of Bax in the treated SGC-7901 cells compared with the controls. The experiment in vivo showed that 0.5, 1, and 2 mg/kg of acetylshikonin significantly inhibited the growth of tumor in the mouse SGC-7901 model, with an inhibitory rate of 25.00%-55.76%. CONCLUSION: Acetylshikonin inhibits the growth of SGC-7901 cells in vitro and in vivo by inducing cell apoptosis. PMID:19370777

[Peripheral mechanisms of action of oxatriazolium-5-olate derivative 3-(3-[1,2,4]-triazolo)oxatriazolium-5-olate in spontaneously hypertensive rats].

PubMed

Artem'eva, M M; Postnikov, A B; Akinfieva, O V; Daliger, I L; Shevelev, S A; Medvedev, O S; Medvedeva, N A

2012-01-01

It is shown that 3-(3-[1,2,4]-triazolo)-oxatriazolium-5-olate (azasidnon-6) can act directly on the vascular wall of isolated segments of caudal ventral artery of SHR rats. Using heme-dependent soluble guanyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), it has been found that one of the possible mechanisms of azasidnon-6 vasodilatory action includes heme-dependent activation of a soluble form of guanylate cyclase.

Contemporary Approaches to Modulating the Nitric Oxide-cGMP Pathway in Cardiovascular Disease.

PubMed

Kraehling, Jan R; Sessa, William C

2017-03-31

Endothelial cells lining the vessel wall control important aspects of vascular homeostasis. In particular, the production of endothelium-derived nitric oxide and activation of soluble guanylate cyclase promotes endothelial quiescence and governs vasomotor function and proportional remodeling of blood vessels. Here, we discuss novel approaches to improve endothelial nitric oxide generation and preserve its bioavailability. We also discuss therapeutic opportunities aimed at activation of soluble guanylate cyclase for multiple cardiovascular indications. © 2017 American Heart Association, Inc.

Modulation of cGMP in Heart Failure

PubMed Central

Boerrigter, Guido; Lapp, Harald; Burnett, John C.

2009-01-01

Heart failure (HF) is a common disease that continues to be associated with high morbidity and mortality warranting novel therapeutic strategies. Cyclic guanosine monophosphate (cGMP) is the second messenger of several important signaling pathways based on distinct guanylate cyclases (GCs) in the cardiovascular system. Both the nitric oxide/soluble GC (NO/sGC) as well as the natriuretic peptide/GC-A (NP/GC-A) systems are disordered in HF, providing a rationale for their therapeutic augmentation. Soluble GC activation with conventional nitrovasodilators has been used for more than a century but is associated with cGMP-independent actions and the development of tolerance, actions which novel NO-independent sGC activators now in clinical development lack. Activation of GC-A by administration of naturally occurring or designer natriuretic peptides is an emerging field, as is the inhibition of enzymes that degrade endogenous NPs. Finally, inhibition of cGMP-degrading phosphodiesterases, particularly phosphodiesterase 5 provides an additional strategy to augment cGMP-signaling. PMID:19089342

[Mechanisms of (2-methyl-n-butyl) shikonin induced apoptosis of gastric cancer SGC-7901 cells].

PubMed

Wang, Hai-Bing; Ma, Xiao-Qiong

2012-06-01

This study is to investigate the effect of (2-methyl-n-butyl) shikonin (MBS) on inducing apoptosis of human gastric cancer cell line SGC-7901 and the role of ERK1/2 signal pathway in the apoptosis. MTT assay was used to detect SGC-7901 cell proliferation. DNA condensation was measured by DAPI stain. Cell apoptosis was analyzed by flow cytometry. Mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. The protein expressions of Bcl-2, Bax, Survivin, cleaved caspase-9, cleaved caspase-3, cleaved PARP, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 and p38 were detected by Western blotting. The results showed that MBS reduced the cell viability of SGC-7901 cells in a dose- and time-dependent manner. The IC50 at 24 h and 48 h for SGC-7901 cells was 10.113 and 4.196 micromolL(-1), respectively. After being treated with MBS, the typical nuclear condensation was observed in SGC-7901 cells by DAPI stain. Apoptosis in SGC-7901 cells was induced by MBS in a dose dependent manner. The protein expression of Bcl-2 was down-regulated, while the protein expressions of cleaved caspase-9, cleaved caspase-3, cleaved PARP, p-ERK1/2 and p-JNK were up-regulated after MBS treatment. U0126, a specific MAP kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by MBS. MMP was decreased by MBS treatment. It can be concluded that MBS could inhibit SGC-7901 cell proliferation and induce apoptosis. Mitochondrial apoptosis pathway, ERK1/2 signal pathway and JNK signal pathway might be involved in this process.

Changes in calcitonin gene-related peptide (CGRP) receptor component and nitric oxide receptor (sGC) immunoreactivity in rat trigeminal ganglion following glyceroltrinitrate pretreatment

PubMed Central

2013-01-01

Background Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN. Methods Isoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling. Results In vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%). Conclusions Prolonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of s

Aspartate 102 in the Heme Domain of Soluble Guanylyl Cyclase Has a Key Role in NO Activation

PubMed Central

Baskaran, Padmamalini; Heckler, Erin J.; van den Akker, Focco; Beuve, Annie

2012-01-01

Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the β subunit of sGC. Still, it is unknown how the breakage of the iron–His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue D102 with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies aspartate 102 (D102) as having a key role in NO activation following breakage of the iron–His bond. PMID:21491881

Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

PubMed

Erdmann, Jeanette; Stark, Klaus; Esslinger, Ulrike B; Rumpf, Philipp Moritz; Koesling, Doris; de Wit, Cor; Kaiser, Frank J; Braunholz, Diana; Medack, Anja; Fischer, Marcus; Zimmermann, Martina E; Tennstedt, Stephanie; Graf, Elisabeth; Eck, Sebastian; Aherrahrou, Zouhair; Nahrstaedt, Janja; Willenborg, Christina; Bruse, Petra; Brænne, Ingrid; Nöthen, Markus M; Hofmann, Per; Braund, Peter S; Mergia, Evanthia; Reinhard, Wibke; Burgdorf, Christof; Schreiber, Stefan; Balmforth, Anthony J; Hall, Alistair S; Bertram, Lars; Steinhagen-Thiessen, Elisabeth; Li, Shu-Chen; März, Winfried; Reilly, Muredach; Kathiresan, Sekar; McPherson, Ruth; Walter, Ulrich; Ott, Jurg; Samani, Nilesh J; Strom, Tim M; Meitinger, Thomas; Hengstenberg, Christian; Schunkert, Heribert

2013-12-19

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Antagonism of soluble guanylyl cyclase attenuates cutaneous vasodilation during whole body heat stress and local warming in humans

PubMed Central

Zhao, Joan L.; Wu, Yubo; Johnson, John M.

2011-01-01

We hypothesized that nitric oxide activation of soluble guanylyl cyclase (sGC) participates in cutaneous vasodilation during whole body heat stress and local skin warming. We examined the effects of the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), on reflex skin blood flow responses to whole body heat stress and on nonreflex responses to increased local skin temperature. Blood flow was monitored by laser-Doppler flowmetry, and blood pressure by Finapres to calculate cutaneous vascular conductance (CVC). Intradermal microdialysis was used to treat one site with 1 mM ODQ in 2% DMSO and Ringer, a second site with 2% DMSO in Ringer, and a third site received Ringer. In protocol 1, after a period of normothermia, whole body heat stress was induced. In protocol 2, local heating units warmed local skin temperature from 34 to 41°C to cause local vasodilation. In protocol 1, in normothermia, CVC did not differ among sites [ODQ, 15 ± 3% maximum CVC (CVCmax); DMSO, 14 ± 3% CVCmax; Ringer, 17 ± 6% CVCmax; P > 0.05]. During heat stress, ODQ attenuated CVC increases (ODQ, 54 ± 4% CVCmax; DMSO, 64 ± 4% CVCmax; Ringer, 63 ± 4% CVCmax; P < 0.05, ODQ vs. DMSO or Ringer). In protocol 2, at 34°C local temperature, CVC did not differ among sites (ODQ, 17 ± 2% CVCmax; DMSO, 18 ± 4% CVCmax; Ringer, 18 ± 3% CVCmax; P > 0.05). ODQ attenuated CVC increases at 41°C local temperature (ODQ, 54 ± 5% CVCmax; DMSO, 86 ± 4% CVCmax; Ringer, 90 ± 2% CVCmax; P < 0.05 ODQ vs. DMSO or Ringer). sGC participates in neurogenic active vasodilation during heat stress and in the local response to direct skin warming. PMID:21292837

Effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells.

PubMed

Sun, Li; Wang, Xu

2003-09-01

To investigate the effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells. The gastric cancer SGC-7901 adenocarcinoma cells were treated with allicin and the cell cycle, inhibitory rate, apoptosis, telomerase activity and morphologic changes were studied by MTT assay, flow cytometry (FCM), TRAP-PCR-ELISA assay, light microscope, electron microscope respectively. Results were compared with that of AZT (3'-Azido-3'-deoxythymidine). SGC-7901 cells were suppressed after exposure to allicin of 0.016 mg/ml, 0.05 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P<0.05). Allicin could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G(2)/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P<0.05). Allicin could inhibit telomerase activity in a time-dependent and dose-dependent pattern. After exposure to allicin at 0.016 mg/ml for 24 hours, SGC-7901 cells showed typical morphologic change. Allicin can inhibit telomerase activity and induce apoptosis of gastric cancer SGC-7901 cells. Allicin may be more effective than AZT.

Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response.

PubMed

Salmon, Didier

2018-04-25

Trypanosoma brucei , etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva ( Salivaria ). In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC) that are topologically similar to receptor-type guanylate cyclase (GC) of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs), rather than the classical protein kinase A cAMP effector (PKA). T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

PubMed

Tremblay, J; Huot, C; Willenbrock, R C; Bayard, F; Gossard, F; Fujio, N; Koch, C; Kuchel, O; Debinski, W; Hamet, P

1993-11-01

Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our

ERK1/2 signalling pathway is involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion.

PubMed

Chen, Liping; Pan, Yuqin; Gu, Ling; Nie, Zhenlin; He, Bangshun; Song, Guoqi; Li, Rui; Xu, Yeqiong; Gao, Tianyi; Wang, Shukui

2013-08-01

This study aimed to investigate the role of CD147 in the progression of gastric cancer and the signalling pathway involved in CD147-mediated gastric cancer cell line SGC7901 proliferation and invasion. Short hairpin RNA (shRNA) expression vectors targeting CD147 were constructed to silence CD147, and the expression of CD147 was monitored by quantitative realtime reverse transcriptase polymerase chain reaction and Western blot and further confirmed by immunohistochemistry in vivo. Cell proliferation was determined by Cell Counting Kit-8 assay, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined by gelatin zymography, and the invasion of SGC7901 was determined by invasion assay. The phosphorylation and non-phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase1/2 (ERK1/2), P38 and c-Jun NH2-terminal kinase were examined by Western blot. Additionally, the ERK1/2 inhibitor U0126 were used to confirm the signalling pathway involved in CD147-mediated SGC7901 progression. The BALB/c nude mice were used to study tumour progression in vivo. The results revealed that CD147 silencing inhibited the proliferation and invasion of SGC7901 cells, and down-regulated the activities of MMP-2 and MMP-9 and the phosphorylation of the ERK1/2 in SGC7901 cells. ERK1/2 inhibitor U0126 decreased the proliferation, and invasion of SGC7901 cells, and down-regulated the MMP-2 and MMP-9 activities. In a nude mouse model of subcutaneous xenografts, the tumour volume was significantly smaller in the SGC7901/shRNA group compared to the SGC7901 and SGC7901/snc-RNA group. Immunohistochemistry analysis showed that CD147 and p-ERK1/2 protein expressions were down-regulated in the SGC7901/shRNA2 group compared to the SGC7901 and SGC7901/snc-RNA group. These results suggest that ERK1/2 pathway involves in CD147-mediated gastric cancer growth and invasion. These findings further highlight the importance of CD147 in cancer progression

SGC Tests for Influence of Material Composition on Compaction Characteristic of Asphalt Mixtures

PubMed Central

Chen, Qun

2013-01-01

Compaction characteristic of the surface layer asphalt mixture (13-type gradation mixture) was studied using Superpave gyratory compactor (SGC) simulative compaction tests. Based on analysis of densification curve of gyratory compaction, influence rules of the contents of mineral aggregates of all sizes and asphalt on compaction characteristic of asphalt mixtures were obtained. SGC Tests show that, for the mixture with a bigger content of asphalt, its density increases faster, that there is an optimal amount of fine aggregates for optimal compaction and that an appropriate amount of mineral powder will improve workability of mixtures, but overmuch mineral powder will make mixtures dry and hard. Conclusions based on SGC tests can provide basis for how to adjust material composition for improving compaction performance of asphalt mixtures, and for the designed asphalt mixture, its compaction performance can be predicted through these conclusions, which also contributes to the choice of compaction schemes. PMID:23818830

SGC tests for influence of material composition on compaction characteristic of asphalt mixtures.

PubMed

Chen, Qun; Li, Yuzhi

2013-01-01

Compaction characteristic of the surface layer asphalt mixture (13-type gradation mixture) was studied using Superpave gyratory compactor (SGC) simulative compaction tests. Based on analysis of densification curve of gyratory compaction, influence rules of the contents of mineral aggregates of all sizes and asphalt on compaction characteristic of asphalt mixtures were obtained. SGC Tests show that, for the mixture with a bigger content of asphalt, its density increases faster, that there is an optimal amount of fine aggregates for optimal compaction and that an appropriate amount of mineral powder will improve workability of mixtures, but overmuch mineral powder will make mixtures dry and hard. Conclusions based on SGC tests can provide basis for how to adjust material composition for improving compaction performance of asphalt mixtures, and for the designed asphalt mixture, its compaction performance can be predicted through these conclusions, which also contributes to the choice of compaction schemes.

8-Nitro-cGMP Attenuates the Interaction between SNARE Complex and Complexin through S-Guanylation of SNAP-25.

PubMed

Kishimoto, Yusuke; Kunieda, Kohei; Kitamura, Atsushi; Kakihana, Yuki; Akaike, Takaaki; Ihara, Hideshi

2018-02-21

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP-25 on the interaction between the SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down assays and coimmunoprecipitation experiments have revealed that S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE complex and cplx. In addition, blue native-PAGE followed by Western blot analysis revealed that the amount of cplx detected at a high molecular weight decreased upon 8-nitro-cGMP treatment in SH-SY5Y cells. These results demonstrated for the first time that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex and cplx.

Curcumin induces apoptosis in SGC-7901 gastric adenocarcinoma cells via regulation of mitochondrial signaling pathways.

PubMed

Xue, Xia; Yu, Jin-Long; Sun, De-Qing; Kong, Feng; Qu, Xian-jun; Zou, Wen; Wu, Jing; Wang, Rong-Mei

2014-01-01

Curcumin, a polyphenol compound derived from the rhizome of the plant Curcuma longa L. has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms by which it induces apoptosis is limited. In this study, the anticancer efficacy of curcumin was investigated in human gastric adenocarcinoma SGC-7901 cells. The results demonstrated that curcumin induced morphological changes and decreased cell viability. Apoptosis triggered by curcumin was visualized using Annexin V-FITC/7- AAD staining. Curcumin-induced apoptosis of SGC-7901 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the down-regulation of Bcl-2 and up-regulation of Bax that led to the cleavage of caspase-3 and increased cleaved PARP was observed in SGC-7901 cells treated with curcumin. Therefore, curcumin-induced apoptosis of SGC-7901 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of curcumin as a potential cancer therapeutic compound.

Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

PubMed Central

Sulakhe, Prakash V.; Narayanan, Njanoor

1978-01-01

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976

DAT-230, a Novel Microtubule Inhibitor, Induced Aberrant Mitosis and Apoptosis in SGC-7901 Cells.

PubMed

Qiao, Foxiao; Zuo, Daiying; Wang, Haifeng; Li, Zengqiang; Qi, Huan; Zhang, Weige; Wu, Yingliang

2013-01-01

2-Methoxy-5-(2-(3,4,5-trimethoxyphenyl)thiophen-3-yl) aniline (DAT-230) is a novel synthesized compound of combretastatin-A-4 derivative with more stability. The present study is to investigate its anti-tumor activity and molecular mechanisms in human gastric adenocarcinoma SGC-7901 cells. DAT-230 inhibited SGC-7901 cells growth. The treatment of DAT-230 resulted in microtubule de-polymerization and G2/M phase arrest. Besides the accumulation and translocation of Cyclin B1, reduction of p-14/15-cdc2 and mitosis delay denoted the Cyclin B1-cdc2 complex active and M phase arrest in SGC-7901 cells treated with DAT-230. Mitochondria pathway participated in apoptosis after G2/M arrest in SGC-7901 cells treated with DAT-230, which was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP), activation of caspase-3 and caspase-9, changes of Bcl-2 and Bax expression, decrease of mitochondrial membrane potential and release of cytochrome c from mitochondria. In vivo, DAT-230 delayed tumor growth in BALB/c nude mice with human gastric adenocarcinoma xenografts. Besides apoptosis was detected with terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay in tumor tissue. In conclusion, DAT-230 is a promising microtubule inhibitor with great anti-tumor activity to SGC-7901, in vitro and in vivo. Its potential to be a candidate of anti-cancer agent is worth of being further investigated.

Ferulic acid-4-O-sulfate rather than ferulic acid relaxes arteries and lowers blood pressure in mice.

PubMed

Van Rymenant, Evelien; Van Camp, John; Pauwels, Bart; Boydens, Charlotte; Vanden Daele, Laura; Beerens, Katrijn; Brouckaert, Peter; Smagghe, Guy; Kerimi, Asimina; Williamson, Gary; Grootaert, Charlotte; Van de Voorde, Johan

2017-06-01

Consumption of foods rich in ferulic acid (FA) such as wholegrain cereals, or FA precursors such as chlorogenic acids in coffee, is inversely correlated with risk of cardiovascular disease and type 2 diabetes. As a result of digestion and phase II metabolism in the gut and liver, FA is converted predominantly into ferulic acid-4-O-sulfate (FA-sul), an abundant plasma metabolite. Although FA-sul is the main metabolite, very little has been reported regarding its bioactivities. We have compared the ex vivo vasorelaxing effect of FA and FA-sul (10 -7 -3.10 -5 M) on isolated mouse arteries mounted in tissue myographs. FA-sul, but not FA, elicited a concentration-dependent vasorelaxation of saphenous and femoral arteries and aortae. The FA-sul-mediated vasorelaxation was blunted by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a soluble guanylate cyclase (sGC) inhibitor. The role of sGC was confirmed in femoral arteries isolated from sGCα 1 (-/-) knockout mice. Furthermore, 4-aminopyridine, a specific inhibitor of voltage-dependent potassium channels, significantly decreased FA-sul-mediated effects. In anesthetized mice, intravenous injection of FA-sul decreased mean arterial pressure, whereas FA had no effect, confirming the results obtained ex vivo. FA-sul is probably one of the major metabolites accounting for the blood pressure-lowering effects associated with FA consumption. Copyright © 2017 Elsevier Inc. All rights reserved.

β,β-Dimethylacrylshikonin Induces Mitochondria Dependent Apoptosis through ERK Pathway in Human Gastric Cancer SGC-7901 Cells

PubMed Central

Ma, Xiao-Qiong; Chen, Jiang-Hua

2012-01-01

β,β-Dimethylacrylshikonin, one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we discussed the molecular mechanisms of β,β-dimethylacrylshikonin in the apoptosis of SGC-7901 cells. β,β-Dimethylacrylshikonin reduced the cell viability of SGC-7901 cells in a dose- and time-dependent manner and induced cell apoptosis. β,β-Dimethylacrylshikonin treatment in SGC-7901 cells down-regulated the expression of XIAP, cIAP-2, and Bcl-2 and up-regulated the expression of Bak and Bax and caused the loss of mitochondrial membrane potential and release of cytochrome c. Additionally, β,β-dimethylacrylshikonin treatment led to activation of caspases-9, 8 and 3, and cleavage of poly (ADP-ribose) polymerase (PARP), which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. β,β-Dimethylacrylshikonin induced phosphorylation of extracellular signal-regulated kinase (ERK) in SGC-7901 cells. U0126, a specific MEK inhibitor, blocked the ERK activation by β,β-dimethylacrylshikonin and abrogated β,β-dimethylacrylshikonin -induced apoptosis. Our results demonstrated that β,β-dimethylacrylshikonin inhibited growth of gastric cancer SGC-7901 cells by inducing ERK signaling pathway, and provided a clue for preclinical and clinical evaluation of β,β-dimethylacrylshikonin for gastric cancer therapy. PMID:22848597

From bedside to bench--meeting report of the 7th International Conference on cGMP "cGMP: generators, effectors and therapeutic implications" in Trier, Germany, from June 19th to 21st 2015.

PubMed

Friebe, Andreas; Sandner, Peter; Seifert, Roland

2015-12-01

During the past decade, our knowledge on the physiology, pathophysiology, basic pharmacology, and clinical pharmacology of the second messenger (cGMP) has increased tremendously. It is now well-established that cGMP, generated by soluble and particulate guanylate cyclases, is highly compartmentalized in cells and regulates numerous body functions. New cGMP-regulated physiological functions include meiosis and temperature perception. cGMP is involved in the genesis of numerous pathologies including cardiovascular, pulmonary, endocrine, metabolic, neuropsychiatric, eye, and tumor diseases. Several new clinical uses of stimulators and activators of soluble guanylate cyclase and of phosphodiesterase inhibitors such as heart failure, kidney failure, cognitive disorders, obesity bronchial asthma, and osteoporosis are emerging. The combination of neprilysin inhibitors-enhancing stimulation of the particulate guanylate cyclase pathway by preventing natriuretic peptide degradation-with angiotensin AT1 receptor antagonists constitutes a novel promising strategy for heart failure treatment. The role of oxidative stress in cGMP signaling, application of cGMP sensors, and gene therapy for degenerative eye diseases are emerging topics. It is anticipated that cGMP research will further prosper over the next years and reach out into more and more basic and clinical disciplines.

Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways.

PubMed

Shen, Yao; Yang, Jianbo; Li, Juan; Shi, Xiaojie; Ouyang, Li; Tian, Yueyang; Lu, Jianxin

2014-01-01

Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

PubMed

Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

2016-09-01

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells.

PubMed

Saxenhofer, H; Fitzgibbon, W R; Paul, R V

1993-02-01

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leitman, D.C.

1988-01-01

The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using {sup 125}I-ANP{sub 8-33}. Specific {sup 125}I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line,more » indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP.« less

Medial prefrontal cortex TRPV1 and CB1 receptors modulate cardiac baroreflex activity by regulating the NMDA receptor/nitric oxide pathway.

PubMed

Lagatta, Davi C; Kuntze, Luciana B; Ferreira-Junior, Nilson C; Resstel, Leonardo B M

2018-05-29

The ventral medial prefrontal cortex (vMPFC) facilitates the cardiac baroreflex response through N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) formation by neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) triggering. Glutamatergic transmission is modulated by the cannabinoid receptor type 1 (CB 1 ) and transient receptor potential vanilloid type 1 (TRPV 1 ) receptors, which may inhibit or stimulate glutamate release in the brain, respectively. Interestingly, vMPFC CB 1 receptors decrease cardiac baroreflex responses, while TRPV 1 channels facilitate them. Therefore, the hypothesis of the present study is that the vMPFC NMDA/NO pathway is regulated by both CB 1 and TRPV 1 receptors in the modulation of cardiac baroreflex activity. In order to test this assumption, we used male Wistar rats that had stainless steel guide cannulae bilaterally implanted in the vMPFC. Subsequently, a catheter was inserted into the femoral artery, for cardiovascular recordings, and into the femoral vein for assessing baroreflex activation. The increase in tachycardic and bradycardic responses observed after the microinjection of a CB 1 receptors antagonist into the vMPFC was prevented by an NMDA antagonist as well as by the nNOS and sGC inhibition. NO extracellular scavenging also abolished these responses. These same pharmacological manipulations inhibited cardiac reflex enhancement induced by TRPV 1 agonist injection into the area. Based on these results, we conclude that vMPFC CB 1 and TRPV 1 receptors inhibit or facilitate the cardiac baroreflex activity by stimulating or blocking the NMDA activation and NO synthesis.

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C.

PubMed

Romi, Hila; Cohen, Idan; Landau, Daniella; Alkrinawi, Suliman; Yerushalmi, Baruch; Hershkovitz, Reli; Newman-Heiman, Nitza; Cutting, Garry R; Ofir, Rivka; Sivan, Sara; Birk, Ohad S

2012-05-04

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms.

PubMed

Qu, Yue; Locock, Katherine; Verma-Gaur, Jiyoti; Hay, Iain D; Meagher, Laurence; Traven, Ana

2016-02-01

Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide β-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms. © The Author 2015. Published by Oxford University Press on behalf of the British

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

Structural and Chemical Biology of Terpenoid Cyclases

PubMed Central

2017-01-01

The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function. PMID:28841019

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

DOE PAGES

Huang, Tingting; Chang, Chin -Yuan; Lohman, Jeremy R.; ...

2016-10-01

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us tomore » propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. In conclusion, these findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.« less

Calmodulin-regulated adenylyl cyclases and neuromodulation.

PubMed

Xia, Z; Storm, D R

1997-06-01

Coincidence detection and crosstalk between signal transduction systems play very important regulatory roles in the nervous system, particularly in the regulation of transcription. Coupling of the Ca2+ and cAMP regulatory systems by calmodulin-regulated adenylyl cyclases is hypothesized to be important for some forms of synaptic plasticity, neuroendocrine function, and olfactory detection. Recent studies of a mutant mouse deficient in type I calmodulin-sensitive adenylyl cyclase have provided the first evidence that adenylyl cyclases are important for synaptic plasticity, as well as for learning and memory in vertebrates.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, G.E.

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.more » 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.« less

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027.

PubMed

Chang, Chin-Yuan; Lohman, Jeremy R; Huang, Tingting; Michalska, Karolina; Bigelow, Lance; Rudolf, Jeffrey D; Jedrzejczak, Robert; Yan, Xiaohui; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2018-03-21

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-β-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

Analysis of erectile responses to BAY 41-8543 and muscarinic receptor stimulation in the rat.

PubMed

Lasker, George F; Pankey, Edward A; Allain, Alexander V; Dhaliwal, Jasdeep S; Stasch, Johannes-Peter; Murthy, Subramanyam N; Kadowitz, Philip J

2013-03-01

Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) and in pathophysiologic conditions where NO formation or bioavailability is impaired, erectile dysfunction (ED) occurs. The aim of this study was to investigate erectile responses to the sGC stimulator BAY 41-8543 in physiologic and pathophysiologic conditions. Increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 41-8543 were investigated in the anesthetized rat. Increases in ICP/MAP in response to ic injections of BAY 41-8543 and the interaction of BAY 41-8543 with exogenous and endogenously released NO were investigated and the effect of the sGC stimulator on cavernosal nerve injury was assessed. The mechanism of the increase in ICP/MAP in response to ic injection of acetylcholine was investigated. The ic injections of BAY 41-8543 increased ICP/MAP and the duration of the response. BAY 41-8543 was less potent than sodium nitroprusside (SNP) and ic injections of BAY 41-8543 and SNP produced a larger response than the algebraic sum of responses to either agent alone. Simultaneous ic injection of BAY 41-8543 and cavernosal nerve stimulation produced a greater response than either intervention alone. Atropine and cavernosal nerve crush injury decreased the response to nerve stimulation and ic injection of BAY 41-8543 restored the response. These data show that BAY 41-8543 has significant erectile activity and can synergize with exogenous and endogenously released NO. This study shows that atropine and nerve crush attenuate the response to cavernosal nerve stimulation and that BAY 41-8543 can restore the response. The results with atropine, L-NAME and hexamethonium indicate that the response to ic injection of acetylcholine is mediated by muscarinic receptors and the release of NO with no significant role for nicotinic receptors. These results suggest that BAY 41-8543 would be useful in the treatment of ED. © 2012 International Society for Sexual

GSNOR Deficiency Enhances In Situ Skeletal Muscle Strength, Fatigue Resistance, and RyR1 S-Nitrosylation Without Impacting Mitochondrial Content and Activity

PubMed Central

Moon, Younghye; Cao, Yenong; Zhu, Jingjing; Xu, Yuanyuan; Balkan, Wayne; Buys, Emmanuel S.; Diaz, Francisca; Kerrick, W. Glenn; Hare, Joshua M.

2017-01-01

Abstract Aim: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. Results: GSNOR null (GSNOR−/−) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR−/− lumbrical muscles showed normal contractility and Ca2+ handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR−/− TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR−/− TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR−/− muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. Innovation: GSNOR may act as a “brake” on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. Conclusions: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165–181. PMID:27412893

Lentiviral Expression of Retinal Guanylate Cyclase-1 (RetGC1) Restores Vision in an Avian Model of Childhood Blindness

PubMed Central

Haire, Shannon E; Aleman, Tomas S; Cideciyan, Artur V; Sokal, Izabel; Palczewski, Krzysztof; Jacobson, Samuel G; Semple-Rowland, Susan L

2006-01-01

Background Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase–1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%–20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. Methods and Findings A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. Conclusions Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness. PMID:16700630

A xanthine-based epithelium-dependent airway relaxant KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) increases respiratory performance and protects against tumor necrosis factor-alpha-induced tracheal contraction, involving nitric oxide release and expression of cGMP and protein kinase G.

PubMed

Lin, Rong-Jyh; Wu, Bin-Nan; Lo, Yi-Ching; An, Li-Mei; Dai, Zen-Kong; Lin, Young-Tso; Tang, Chao-Shun; Chen, Ing-Jun

2006-02-01

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.

ADP-ribosyl cyclases regulate early development of the sea urchin.

PubMed

Ramakrishnan, Latha; Uhlinger, Kevin; Dale, Leslie; Hamdoun, Amro; Patel, Sandip

2016-06-01

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca 2+ signalling such as cADPR and NAADP. Although Ca 2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca 2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

Effect of Vericiguat, a Soluble Guanylate Cyclase Stimulator, on Natriuretic Peptide Levels in Patients With Worsening Chronic Heart Failure and Reduced Ejection Fraction: The SOCRATES-REDUCED Randomized Trial.

PubMed

Gheorghiade, Mihai; Greene, Stephen J; Butler, Javed; Filippatos, Gerasimos; Lam, Carolyn S P; Maggioni, Aldo P; Ponikowski, Piotr; Shah, Sanjiv J; Solomon, Scott D; Kraigher-Krainer, Elisabeth; Samano, Eliana T; Müller, Katharina; Roessig, Lothar; Pieske, Burkert

2015-12-01

Worsening chronic heart failure (HF) is a major public health problem. To determine the optimal dose and tolerability of vericiguat, a soluble guanylate cyclase stimulator, in patients with worsening chronic HF and reduced left ventricular ejection fraction (LVEF). Dose-finding phase 2 study that randomized 456 patients across Europe, North America, and Asia between November 2013 and January 2015, with follow-up ending June 2015. Patients were clinically stable with LVEF less than 45% within 4 weeks of a worsening chronic HF event, defined as worsening signs and symptoms of congestion and elevated natriuretic peptide level requiring hospitalization or outpatient intravenous diuretic. Placebo (n = 92) or 1 of 4 daily target doses of oral vericiguat (1.25 mg [n = 91], 2.5 mg [n = 91], 5 mg [n = 91], 10 mg [n = 91]) for 12 weeks. The primary end point was change from baseline to week 12 in log-transformed level of N-terminal pro-B-type natriuretic peptide (NT-proBNP). The primary analysis specified pooled comparison of the 3 highest-dose vericiguat groups with placebo, and secondary analysis evaluated a dose-response relationship with vericiguat and the primary end point. Overall, 351 patients (77.0%) completed treatment with the study drug with valid 12-week NT-proBNP levels and no major protocol deviation and were eligible for primary end point evaluation. In primary analysis, change in log-transformed NT-proBNP levels from baseline to week 12 was not significantly different between the pooled vericiguat group (log-transformed: baseline, 7.969; 12 weeks, 7.567; difference, -0.402; geometric means: baseline, 2890 pg/mL; 12 weeks, 1932 pg/mL) and placebo (log-transformed: baseline, 8.283; 12 weeks, 8.002; difference, -0.280; geometric means: baseline, 3955 pg/mL; 12 weeks, 2988 pg/mL) (difference of means, -0.122; 90% CI, -0.32 to 0.07; ratio of geometric means, 0.885, 90% CI, 0.73-1.08; P = .15). The exploratory secondary analysis suggested a

Systemic sclerosis and localized scleroderma--current concepts and novel targets for therapy.

PubMed

Distler, Oliver; Cozzio, Antonio

2016-01-01

Systemic sclerosis (SSc) is a chronic autoimmune disease with a high morbidity and mortality. Skin and organ fibrosis are key manifestations of SSc, for which no generally accepted therapy is available. Thus, there is a high unmet need for novel anti-fibrotic therapeutic strategies in SSc. At the same time, important progress has been made in the identification and characterization of potential molecular targets in fibrotic diseases over the recent years. In this review, we have selected four targeted therapies, which are tested in clinical trials in SSc, for in depths discussion of their preclinical characterization. Soluble guanylate cyclase (sGC) stimulators such as riociguat might target both vascular remodeling and tissue fibrosis. Blockade of interleukin-6 might be particularly promising for early inflammatory stages of SSc. Inhibition of serotonin receptor 2b signaling links platelet activation to tissue fibrosis. Targeting simultaneously multiple key molecules with the multityrosine kinase-inhibitor nintedanib might be a promising approach in complex fibrotic diseases such as SSc, in which many partially independent pathways are activated. Herein, we also give a state of the art overview of the current classification, clinical presentation, diagnostic approach, and treatment options of localized scleroderma. Finally, we discuss whether the novel targeted therapies currently tested in SSc could be used for localized scleroderma.

Effects of Lubrol detergents on adenylate cyclases.

PubMed

Bär, H P; Kulshrestha, S

1975-04-01

The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects onbasal and stimulated adenylate cyclases. Above concentrations of 0.001-0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibed about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer centain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

PubMed Central

Maresca, Julia A.; Graham, Joel E.; Wu, Martin; Eisen, Jonathan A.; Bryant, Donald A.

2007-01-01

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria. PMID:17606904

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

PubMed

Maresca, Julia A; Graham, Joel E; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A

2007-07-10

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

DOE PAGES

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan; ...

2017-08-30

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway.

PubMed

Lin, Ruhui; Li, Zuanfang; Lin, Jiumao; Ye, Jinxia; Cai, Qiaoyan; Chen, Lidian; Peng, Jun

2015-10-01

Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis.

Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway

PubMed Central

LIN, RUHUI; LI, ZUANFANG; LIN, JIUMAO; YE, JINXIA; CAI, QIAOYAN; CHEN, LIDIAN; PENG, JUN

2015-01-01

Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis. PMID:26622854

Preservation of endothelium-dependent relaxation in atherosclerotic mice with endothelium-restricted endothelin-1 overexpression.

PubMed

Mian, Muhammad Oneeb Rehman; Idris-Khodja, Noureddine; Li, Melissa W; Leibowitz, Avshalom; Paradis, Pierre; Rautureau, Yohann; Schiffrin, Ernesto L

2013-10-01

In human atherosclerosis, which is associated with elevated plasma and coronary endothelin (ET)-1 levels, ETA receptor antagonists improve coronary endothelial function. Mice overexpressing ET-1 specifically in the endothelium (eET-1) crossed with atherosclerosis-prone apolipoprotein E knockout mice (Apoe(-/-)) exhibit exaggerated high-fat diet (HFD)-induced atherosclerosis. Since endothelial dysfunction often precedes atherosclerosis development, we hypothesized that mice overexpressing endothelial ET-1 on a genetic background deficient in apolipoprotein E (eET-1/Apoe(-/-)) would have severe endothelial dysfunction. To test this hypothesis, we investigated endothelium-dependent relaxation (EDR) to acetylcholine in eET-1/Apoe(-/-) mice. EDR in mesenteric resistance arteries from 8- and 16-week-old mice fed a normal diet or HFD was improved in eET-1/Apoe(-/-) compared with Apoe(-/-) mice. Nitric oxide synthase (NOS) inhibition abolished EDR in Apoe(-/-). EDR in eET-1/Apoe(-/-) mice was resistant to NOS inhibition irrespective of age or diet. Inhibition of cyclooxygenase, the cytochrome P450 pathway, and endothelium-dependent hyperpolarization (EDH) resulted in little or no inhibition of EDR in eET-1/Apoe(-/-) compared with wild-type (WT) mice. In eET-1/Apoe(-/-) mice, blocking of EDH or soluble guanylate cyclase (sGC), in addition to NOS inhibition, decreased EDR by 36 and 30%, respectively. The activation of 4-aminopyridine-sensitive voltage-dependent potassium channels (Kv) during EDR was increased in eET-1/Apoe(-/-) compared with WT mice. We conclude that increasing eET-1 in mice that develop atherosclerosis results in decreased mutual dependence of endothelial signaling pathways responsible for EDR, and that NOS-independent activation of sGC and increased activation of Kv are responsible for enhanced EDR in this model of atherosclerosis associated with elevated endothelial and circulating ET-1.

Nitric oxide augments single Ca(2+) channel currents via cGMP-dependent protein kinase in Kenyon cells isolated from the mushroom body of the cricket brain.

PubMed

Kosakai, Kumiko; Tsujiuchi, Yuuki; Yoshino, Masami

2015-07-01

Behavioral and pharmacological studies in insects have suggested that the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is involved in the formation of long-term memory (LTM) associated with olfactory learning. However, the target molecules of NO and the downstream signaling pathway are still not known. In this study, we investigated the action of NO on single voltage-dependent Ca(2+) channels in the intrinsic neurons known as Kenyon cells within the mushroom body of the cricket brain, using the cell-attached configuration of the patch-clamp technique. Application of the NO donor S-nitrosoglutathione (GSNO) increased the open probability (NPO) of single Ca(2+) channel currents. This GSNO-induced increase was blocked by ODQ, a soluble guanylate cyclase (sGC) inhibitor, suggesting that the NO generated by GSNO acts via sGC to raise cGMP levels. The membrane-permeable cGMP analog 8-Bro-cGMP also increased the NPO of single Ca(2+) channel currents. Pretreatment of cells with KT5823, a protein kinase G blocker, abolished the excitatory effect of GSNO. These results suggest that NO augments the activity of single Ca(2+) channels via the cGMP/PKG signaling pathway. To gain insight into the physiological role of NO, we examined the effect of GSNO on action potentials of Kenyon cells under current-clamp conditions. Application of GSNO increased the frequency of action potentials elicited by depolarizing current injections, indicating that NO acts as a modulator resulting in a stimulatory signal in Kenyon cells. We discuss the increased Ca(2+) influx through these Ca(2+) channels via the NO/cGMP signaling cascade in relation to the formation of olfactory LTM. Copyright © 2015 Elsevier Ltd. All rights reserved.

The guanylyl cyclase family at Y2K.

PubMed

Wedel, B; Garbers, D

2001-01-01

During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

Adenylyl cyclase and G-proteins in Phytomonas.

PubMed

Farber, M D; Montagna, A E; Paveto, C; Dollet, M; Sanchex-Moreno, M; Osuna, A; Torres, H N; Flawia, M M

1995-01-01

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

PubMed

Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

2013-06-01

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

Pituitary adenylate cyclase-activating polypeptide: a novel peptide with protean implications.

PubMed

Pisegna, Joseph R; Oh, David S

2007-02-01

The purpose of this review is to highlight the importance of pituitary adenylate cyclase-activating polypeptide in physiological processes and to describe how this peptide is becoming increasingly recognized as having a major role in the body. Since its discovery in 1989, investigators have sought to determine the site of biological activity and the function of pituitary adenylate cyclase-activating polypeptide in maintaining homeostasis. Since its discovery, pituitary adenylate cyclase-activating polypeptide appears to play an important role in the regulation of processes within the central nervous system and gastrointestinal tract, as well in reproductive biology. Pituitary adenylate cyclase-activating polypeptide has been shown to regulate tumor cell growth and to regulate immune function through its effects on T lympocytes. These discoveries suggest the importance of pituitary adenylate cyclase-activating polypeptide in neuronal development, neuronal function, gastrointestinal tract function and reproduction. Future studies will examine more closely the role of pituitary adenylate cyclase-activating polypeptide in regulation of malignantly transformed cells, as well as in regulation of immune function.

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

Regulation of NANC neural bronchoconstriction in vivo in the guinea-pig: involvement of nitric oxide, vasoactive intestinal peptide and soluble guanylyl cyclase.

PubMed

Lei, Y H; Barnes, P J; Rogers, D F

1993-01-01

1. We investigated the effect of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and the peptidase alpha-chymotrypsin on non-adrenergic, non-cholinergic (NANC neural) bronchoconstriction induced by electrical stimulation of the vagus nerves and by capsaicin in anaesthetized guinea-pigs in vivo using pulmonary insufflation pressure (PIP) as an index of bronchial tone. We also investigated the contribution of soluble guanylyl cyclase (SGC) to NANC neural relaxant mechanisms. 2. In the presence of atropine and propranolol, electrical stimulation of the vagus nerves induced a frequency-dependent increase in PIP above baseline of 67% at 2.5 Hz, of 128% at 5 Hz and of 230% at 10 Hz. L-NAME (1-50 mg kg-1, i.v.), at doses inducing increases in systemic blood pressure, dose-relatedly potentiated NANC bronchoconstriction. At 10 mg kg-1 i.v., L-NAME significantly (P < 0.05) potentiated NANC bronchoconstriction by a further 106% at 2.5 Hz and a further 147% at 5 Hz but did not potentiate the increase in PIP at 10 Hz. L-NAME did not induce bronchoconstriction in sham-stimulated control animals. D-NAME did not potentiate NANC bronchoconstriction. Raising systemic blood pressure with phenylephrine did not potentiate vagally-induced bronchoconstriction (2.5 Hz). 3. The NO precursor L-arginine, but not D-arginine, (100 mg kg-1, i.v.) significantly reversed the potentiation by L-NAME of NANC bronchoconstriction. L-Arginine alone significantly inhibited neurogenic bronchoconstriction at 10 Hz (by 74%); the inhibition of 25% at 2.5 Hz was not significant. 4. L-NAME did not significantly affect the increases in PIP induced by intravenous substance P. neurokinin A (NKA) or capsaicin. 5. The inhibitor of SGC, methylene blue (10 mg kg', i.v.) potentiated (by 110-140%) NANC neural bronchoconstriction induced by lower frequencies of nerve stimulation and reversed the reduction in PIP induced by the SGC activator, sodium nitroprusside (SNP, 1.05 mg kg- 1, i

Participation of hippocampal nitric oxide synthase and soluble guanylate cyclase in the modulation of behavioral responses elicited by the rat forced swimming test.

PubMed

Sales, Amanda J; Hiroaki-Sato, Vinícius A; Joca, Sâmia R L

2017-02-01

Systemic or hippocampal administration of nitric oxide (NO) synthase inhibitors induces antidepressant-like effects in animals, implicating increased hippocampal levels of NO in the neurobiology of depression. However, the role played by different NO synthase in this process has not been clearly defined. As stress is able to induce neuroinflammatory mechanisms and trigger the expression of inducible nitric oxide synthase (iNOS) in the brain, as well as upregulate neuronal nitric oxide synthase (nNOS) activity, the aim of the present study was to investigate the possible differential contribution of hippocampal iNOS and nNOS in the modulation of the consequences of stress elicited by the forced swimming test. Male Wistar rats received intrahippocampal injections, immediately after the pretest or 1 h before the forced swimming test, of selective inhibitors of nNOS (N-propyl-L-arginine), iNOS (1400W), or sGC (ODQ), the main pharmacological target for NO. Stress exposure increased nNOS and phospho-nNOS levels at all time points, whereas iNOS expression was increased only 24 h after the pretest. All drugs induced an antidepressant-like effect. However, whereas the nNOS inhibitor was equally effective when injected at different times, the iNOS inhibitor was more effective 24 h after the pretest. These results suggest that hippocampal nNOS and iNOS contribute to increase in NO levels in response to stress, although with a differential time course after stress exposure.

Refractoriness of the gravid rat uterus to tocolytic and biochemical effects of atrial natriuretic peptide.

PubMed Central

Potvin, W.; Varma, D. R.

1990-01-01

1. Effects of atrial natriuretic peptide (ANP) on tension development, particulate guanylate cyclase activity and guanosine 3':5'-cyclic monophosphate (cyclic GMP) concentrations of uteri from oestrogen-treated, progesterone-treated, ovariectomized and pregnant rats were determined in vitro. 2. ANP inhibited the tension development by myometrial tissues from oestrogen-treated virgin rats and the sterile horn of 10 to 14 day pregnant rats but not of the uterus from pregnant and progesterone-treated rats. 3. Inhibition of cyclo-oxygenase and lipoxygenase activities did not restore the tocolytic activity of ANP on gravid uterus. ANP exerted a tocolytic effect on nongravid uterus submaximally stimulated by prostaglandin F2 alpha (PGF2 alpha), oxytocin, vasopressin, angiotensin II or 5-hydroxytryptamine (5-HT). 4. Ovariectomy decreased the tocolytic effects of ANP, which could be restored by oestrogen treatment. 5. The refractoriness to the tocolytic effect of ANP in pregnant rats was not accompanied by a decrease in its relaxant effects on isolated aortic strips. 6. Tocolytic effects of isoprenaline, isobutylmethyl xanthine and hydroxylamine were not influenced by pregnancy or progesterone treatment. Up to a concentration of 3 mM, sodium nitroprusside did not affect myometrial tension development. 7. Pregnancy and progesterone treatment markedly inhibited ANP-induced increases in myometrial particulate guanylate cyclase activity and cyclic GMP concentrations but did not influence the effects of ANP on aortic cyclic GMP concentrations. 8. It is concluded that exposure of the myometrium to circulating and placentally-produced progesterone is responsible for the pregnancy-induced decrease in the effects of ANP on myometrial particulate guanylate cyclase activity and cyclic GMP concentrations and in turn on myometrial tension development. PMID:1974161

Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development

PubMed Central

Becker, Jason R.; Chatterjee, Sneha; Robinson, Tamara Y.; Bennett, Jeffrey S.; Panáková, Daniela; Galindo, Cristi L.; Zhong, Lin; Shin, Jordan T.; Coy, Shannon M.; Kelly, Amy E.; Roden, Dan M.; Lim, Chee Chew; MacRae, Calum A.

2014-01-01

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways. PMID:24353062

ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia.

PubMed

Chau, Y P; Lu, K S

1994-10-01

Cytochemical relationship between Golgi complex and dense-cored granules (DCGs) of small granule-containing (SGC) cells in rat superior cervical ganglia was examined in electron microscopy by zinc-iodide-osmium tetroxide (ZIO) method and by enzyme cytochemistry for thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). After ZIO impregnation, all the saccules of Golgi apparatus and some of tubular rough endoplasmic reticulum (rER) were stained. DCGs in periphery of SGC cells were not stained, but varying degrees of dense deposits occurred in the DCGs in vicinity of Golgi trans-saccules. Both TPPase and ACPase activities were localized in one or two stacked layers of saccules on the trans side of the Golgi complex. No reaction products were demonstrated in the DCGs. From these results, we suggest that the DCGs of SGC cells in rat superior cervical ganglia are derived from the Golgi complex, and that lysosomal cleavage of protein contents in the DCGs may occur in the trans Golgi saccules.

Bordetella Adenylate Cyclase-Hemolysin Toxins

PubMed Central

Guiso, Nicole

2017-01-01

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

Effects of a Shuangling Fuzheng anticancer preparation on the proliferation of SGC-7901 cells and immune function in a cyclophosphamide-treated murine model.

PubMed

Chen, Hua-Sheng; Chen, Jue; Cui, De-Li; Zheng, Yuan-Yuan; Xu, Ai-Hua; Chen, Gang; Jia, Ling-Chang

2007-12-28

To study the inhibitory effects of a Shuangling Fuzheng anticancer preparation (SFAP) on the human gastric cancer cell line SGC-7901 in vitro as well as its immune-modulated effects in a cyclophosphamide-treated murine model. MTT experiments and immunocytochemistry ABC experiments were performed for detecting the proliferation of SGC-7901 cells in vitro and protein expression of c-myc. The staphylococcal protein A (SPA) rosette test was utilized for measuring the ratio of T-lymphocyte subsets from peripheral blood in a cyclophosphamide-treated murine model. Enzyme-linked immunosorbant assay (ELISA) was performed for measuring the levels of serum sIL-2R in treated mice, while immunoturbidimetry was used for measuring the levels of immunoglobulins (Ig). SFAP (40-640 mg/L, 48 h) inhibited the proliferation of SGC-7901 cells, and a positive correlation was noted between inhibitory effects and dosage. At a dosage of 160-320 mg/L in cultured cells, the expression of c-myc was decreased. SFAP (50-200 mg/kg) increased the percentage of CD3+ and CD4+ T-lymphocytes, the ratio of CD4/CD8, and the contents of Ig such as IgM, IgG or IgA, but decreased the levels of serum sIL-2R in peripheral blood from cyclophosphamide-treated mice. SFAP can inhibit the proliferation of SGC-7901 cells via the c-myc gene. In addition, SFAP can modulat the cellular and humoral immunity in cyclophosphamide-induced immunosuppressed mice.

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.

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

2015-08-07

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). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Adenylate cyclase from rabbit heart: substrate binding site].

PubMed

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

PubMed Central

Bitensky, M W; Wheeler, M A; Mehta, H; Miki, N

1975-01-01

Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from, and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD. PMID:1058474

Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

PubMed

Lu, Wan-Jung; Chang, Nen-Chung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Chou, Duen-Suey; Thomas, Philip Aloysius; Sheu, Joen-Rong

2014-12-01

CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

PubMed

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Modification of adenylate cyclase by photoaffinity analogs of forskolin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, L.T.; Nie, Z.M.; Mende, T.J.

1989-01-01

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography followingmore » SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.« less

Heme-based sensors in biological systems.

PubMed

Rodgers, K R

1999-04-01

The past several years have been witness to a staggering rate of advancement in the understanding of how organisms respond to changes in the availability of diatomic molecules that are toxic and/or crucial to survival. Heme-based sensors presently constitute the majority of the proteins known to sense NO, O2 and CO and to initiate the chemistry required to adapt to changes in their availabilities. Knowledge of the three characterized members of this class, soluble guanylate cyclase, FixL and CooA, has grown substantially during the past year. The major advances have resulted from a broad range of approaches to elucidation of both function and mechanism. They include growth in the understanding of the interplay between the heme and protein in soluble guanylate cyclase, as well as alternate means for its stimulation. Insight into the O2-induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum. Finally, the ligation environment and ligand interchange that facilitates CO sensing by CooA has been established by spectroscopic and mutagenesis techniques.

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

PubMed Central

Yoshimura, M; Cooper, D M

1992-01-01

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+. Images PMID:1379717

Downregulated expression of the cyclase-associated protein 1 (CAP1) reduces migration in esophageal squamous cell carcinoma.

PubMed

Li, Mei; Yang, Xiaojing; Shi, Hui; Ren, Hanru; Chen, Xueyu; Zhang, Shu; Zhu, Junya; Zhang, Jianguo

2013-09-01

Overexpression of cyclase-associated proteins has been associated with poor prognosis in several human cancers. Cyclase-associated protein 1 is a member of the cyclase-associated proteins which contributes to tumor progression. The aim of the present study was to examine the expression of cyclase-associated protein 1 and to elucidate its clinicopathologic significance in a larger series of esophageal squamous cell carcinoma. Immunohistochemical and western blot analyses were performed in esophageal squamous cell carcinoma tissues. Survival analyses were performed by using the Kaplan-Meier method. The role of cyclase-associated protein 1 in migration was studied in esophageal squamous cell carcinoma cell lines of TE1 through knocking down cyclase-associated protein 1 with siRNA and overexpression of cyclase-associated protein 1. The regulation of cyclase-associated protein 1 on migration was determined by transwell and wound-healing assays. Immunohistochemical analysis showed that cyclase-associated protein 1 expression was negatively associated with E-cadherin and significantly associated with lymph node metastases. Survival analysis revealed that cyclase-associated protein 1 overexpression was significantly associated with overall survival (P = 0.011). Knock down of cyclase-associated protein 1 in TE1 cells resulted in decreased vimentin and F-actin levels and the capability for migration. In addition, overexpression of cyclase-associated protein 1 promoted the migration of TE1 cells. These findings suggest that cyclase-associated protein 1 is involved in the metastasis of esophageal squamous cell carcinoma, and that elevated levels of cyclase-associated protein 1 expression may indicate a poor prognosis for patients with esophageal squamous cell carcinoma.

(1)H, (13)C, (15)N backbone and side-chain resonance assignment of Nostoc sp. C139A variant of the heme-nitric oxide/oxygen binding (H-NOX) domain.

PubMed

Alexandropoulos, Ioannis I; Argyriou, Aikaterini I; Marousis, Kostas D; Topouzis, Stavros; Papapetropoulos, Andreas; Spyroulias, Georgios A

2016-10-01

The H-NOX (Heme-nitric oxide/oxygen binding) domain is conserved across eukaryotes and bacteria. In human soluble guanylyl cyclase (sGC) the H-NOX domain functions as a sensor for the gaseous signaling agent nitric oxide (NO). sGC contains the heme-binding H-NOX domain at its N-terminus, which regulates the catalytic site contained within the C-terminal end of the enzyme catalyzing the conversion of GTP (guanosine 5'-triphosphate) to GMP (guanylyl monophosphate). Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 183-residue H-NOX domain from Nostoc sp. through solution NMR.

General Base-General Acid Catalysis by Terpenoid Cyclases§

PubMed Central

Pemberton, Travis A.; Christianson, David W.

2016-01-01

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. PMID:27072285

Effect of age and posture on human lymphocyte adenylate cyclase activity.

PubMed

Mader, S L; Robbins, A S; Rubenstein, L Z; Tuck, M L; Scarpace, P J

1988-03-01

1. A number of age-related changes have been reported in the catecholamine-adrenoceptor-adenylate cyclase system. Most of the data available on these alterations come from resting subjects; the response to acute stress may provide additional insights into the age effect on these responses. 2. We measured supine and 10 min upright plasma noradrenaline and lymphocyte adenylate cyclase activity in ten healthy elderly subjects (age 66-80 years) and seven healthy young subjects (age 27-34 years). 3. Isoprenaline stimulation of lymphocyte adenylate cyclase activity was not significantly different between supine and upright positions or between elderly and young subjects. There was a marked increase in forskolin-stimulated adenylate cyclase activity in the upright posture in both elderly and young subjects. The increment over supine levels was 70% in the elderly (P less than 0.025) and 73% in the young (P less than 0.05). This enhanced forskolin activity was not seen in two young subjects who became syncopal. 4. These data suggest that enhanced forskolin-stimulated adenylate cyclase activity occurs after 10 min of upright posture in both elderly and young subjects, and may be relevant to immediate blood pressure regulation. We were unable to demonstrate any age-related differences in these acute adrenergic responses.

Magnolol, a natural compound, induces apoptosis of SGC-7901 human gastric adenocarcinoma cells via the mitochondrial and PI3K/Akt signaling pathways.

PubMed

Rasul, Azhar; Yu, Bo; Khan, Muhammad; Zhang, Kun; Iqbal, Furhan; Ma, Tonghui; Yang, Hong

2012-04-01

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies.

Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

PubMed

Zan, Xinyi; Cui, Fengjie; Li, Yunhong; Yang, Yan; Wu, Di; Sun, Wenjing; Ping, Lifeng

2015-05-01

HEG-5 is a novel polysaccharide-protein purified from the fermented mycelia of Hericium erinaceus CZ-2. The present study aims to investigate the effects of HEG-5 on proliferation, cell cycle and apoptosis of human gastric cancer cells SGC-7901. Here, we first uncover that HEG-5 significantly inhibited the proliferation and colony formation of SGC-7901 cells by promoting apoptosis and cell cycle arrest at S phase. RT-PCR and Western blot analysis suggested that HEG-5 could decrease the expressions of Bcl2, PI3K and AKT1, while increase the expressions of Caspase-8, Caspase-3, p53, CDK4, Bax and Bad. These findings indicated that the Caspase-8/-3-dependent, p53-dependent mitochondrial-mediated and PI3k/Akt signaling pathways involved in the molecular events of HEG-5 induced apoptosis and cell cycle arrest. Thus, our study provides in vitro evidence that HEG-5 may be taken as a potential candidate for treating gastric cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masure, H.R.; Donovan, M.G.; Storm, D.R.

1991-01-01

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increasesmore » in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.« less

The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shuai; Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing; Zou, Lihui

Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured humanmore » PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension.« less

Epigallocatechin-3-gallate suppressed the over-expression of HSP 70 and MDR1 induced by heat shock in SGC 7901.

PubMed

Tang, Xiao-Yan; Zhu, You-Qing

2008-06-01

This study investigated the effects of epigallocatechin-3-gallate (EGCG) on the expression of HSP 70 and MDR 1. SGC-7901 cells were cultured with RPMI 1640 medium. The single or combined effects of EGCG (0.1, 1, 10, 20, and 40 micromol/L) and heat shock were examined by MTT assay. The expression of HSP 70 and MDR 1 was semi-quantified by the reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry method (SP staining). EGCG suppressed cell proliferation at a time- and dose-dependent manner. The effects of combined treatment with EGCG and heat shock on the growth of SGC-7901 cells were stronger than single effects of EGCG. After using EGCG for 24 h, 48 h and 72 h, the IC50s were 112.5 micromol/l, 21.41 micromol/l and 5.24 micromol/l, respectively. Heat shock stimulated the over-expression of HSP 70, especially after heat shock for 8 h, as well as MDR1 after heat shock for 24 h. But EGCG suppressed the over-expression induced by heat shock. The authors conclude that EGCG inhibited the proliferation of SGC-7901, and EGCG combined with heat shock strengthened the effects. Heat shock weakened the over-expression of HSP 70 and MDR1; however, EGCG suppressed the over-expression of HSP 70 and MDR1 induced by heat shock. EGCG combined with heat shock may enhance the sensitivity of drugs to tumors.

Neuroprotective potential of high-dose biotin.

PubMed

McCarty, Mark F; DiNicolantonio, James J

2017-11-01

A recent controlled trial has established that high-dose biotin supplementation - 100 mg, three times daily - has a stabilizing effect on progression of multiple sclerosis (MS). Although this effect has been attributed to an optimization of biotin's essential cofactor role in the brain, a case can be made that direct stimulation of soluble guanylate cyclase (sGC) by pharmacological concentrations of biotin plays a key role in this regard. The utility of high-dose biotin in MS might reflect an anti-inflammatory effect of cGMP on the cerebral microvasculature, as well on oligodendrocyte differentiation and on Schwann cell production of neurotrophic factors thought to have potential for managing MS. But biotin's ability to boost cGMP synthesis in the brain may have broader neuroprotective potential. In many types of neurons and neural cells, cGMP exerts neurotrophic-mimetic effects - entailing activation of the PI3K-Akt and Ras-ERK pathways - that promote neuron survival and plasticity. Hippocampal long term potentiation requires nitric oxide synthesis, which in turn promotes an activating phosphorylation of CREB via a pathway involving cGMP and protein kinase G (PKG). In Alzheimer's disease (AD), amyloid beta suppresses this mechanism by inhibiting sGC activity; agents which exert a countervailing effect by boosting cGMP levels tend to restore effective long-term potentiation in rodent models of AD. Moreover, NO/cGMP suppresses amyloid beta production within the brain by inhibiting expression of amyloid precursor protein and BACE1. In conjunction with cGMP's ability to oppose neuron apoptosis, these effects suggest that high-dose biotin might have potential for the prevention and management of AD. cGMP also promotes neurogenesis, and may lessen stroke risk by impeding atherogenesis and hypertrophic remodeling in the cerebral vasculature. The neuroprotective potential of high-dose biotin likely could be boosted by concurrent administration of brain

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

Magnolol, a natural compound, induces apoptosis of SGC-7901 human gastric adenocarcinoma cells via the mitochondrial and PI3K/Akt signaling pathways

PubMed Central

RASUL, AZHAR; YU, BO; KHAN, MUHAMMAD; ZHANG, KUN; IQBAL, FURHAN; MA, TONGHUI; YANG, HONG

2012-01-01

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies. PMID:22139054

A randomized, controlled phase I/II study to evaluate the safety and efficacy of MGV354 for ocular hypertension or glaucoma.

PubMed

Stacy, Rebecca; Huttner, Kenneth; Watts, Jen; Peace, James; Wirta, David; Walters, Tom; Sall, Kenneth; Seaman, John; Ni, Xiao; Prasanna, Ganesh; Mogi, Muneto; Adams, Christopher; Yan, Jing-He; Wald, Michael; He, Yunsheng; Newton, Ronald; Kolega, Randall; Grosskreutz, Cynthia

2018-05-23

To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma DESIGN: Double-masked, randomized, and vehicle-controlled study METHODS: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site. Part 3 was a multi-site trial that evaluated IOP-lowering efficacy of the MTD administered nightly for one week in 50 patients with minimum IOP of 24mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at Day 8 compared to baseline (ClinicalTrials.gov NCT02743780). There was no difference in favor of MGV354 for IOP lowering; change from Baseline to Day 8 in mean diurnal IOP was -0.6 mmHg for MGV354-treated patients and -1.1 mmHg for Vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common adverse events reported after MGV354 administration were conjunctival and ocular hyperemia. Overall, MGV354 0.1% demonstrated no statistically significant effect compared to Vehicle in lowering IOP based upon the study's main outcome measure. MGV354 produced ocular hyperemia consistent with its pharmacology. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Inositol uptake in rat aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl

1990-01-01

{sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositolmore » uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.« less

Comparison of the effects of histamine and tolazoline on adenylate cyclase activity from guinea pig heart.

PubMed

Weinryb, I; Michel, I M

1975-01-01

Both histamine and tolazoline (2-benzyl-2-imidazoline) stimulated particulate fractions of adenylate cyclase from guinea pig myocardium. Tolazoline was one-tenth as potent, and about two-thirds as active at maximally effective levels, as was histamine. Enhancement of cyclase activity by tolazoline was additive with that by isoproterenol, and the histamine and tolazoline concentration-response curves were parallel, suggesting that tolazoline acted at the same site as histamine. At maximally effective concentrations, tolazoline did not affect ATPase or cyclic AMP phosphodiesterase activities associated with the cyclase preparations. The H1-receptor antagonist mepyramine, and the H2 antagonist, burimamide, blocked stimulation of cyclase by tolazoline at one-tenth the molarity of agonist. Both antagonists were less effective vs. histamine stimulation of heart cyclase in particulate fractions or whole homogenates, with mepyramine being generally more potent. It is suggested that the molecular basis of the stimulatory effect of tolazoline on cardiac tissue may be histaminergic stimulation of adenylate cyclase. Furthermore, the lack of potency of burimamide as a histamine antagonist and its lack of specificity compared to mepyramine, at the subcellular level, indicate that histamine-responsive adenylate cyclase from heart may not be a satisfactory molecular model for the H2 receptor pharmacology of histamine in cardiac tissue.

Action of Escherichia coli Enterotoxin: Adenylate Cyclase Behavior of Intestinal Epithelial Cells in Culture

PubMed Central

Kantor, Harvey S.; Tao, Pearl; Wisdom, Charlene

1974-01-01

Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model. PMID:4364505

Derivatizations of Sgc8-c aptamer to prepare metallic radiopharmaceuticals as imaging diagnostic agents: Syntheses, isolations, and physicochemical characterizations.

PubMed

Sicco, Estefanía; Báez, Jessica; Margenat, Jimena; García, Fernanda; Ibarra, Manuel; Cabral, Pablo; Moreno, María; Cerecetto, Hugo; Calzada, Victoria

2018-03-01

Aptamers, oligonucleotides with the capability to bind to a target through non-covalent bonds with high affinity and specificity, have a great number of advantages as scaffold to prepare molecular imaging agents. In this sense, we have performed post-SELEX modifications of a truncated aptamer, Sgc8-c, which bind to protein tyrosine kinase 7 to obtain a specific molecular targeting probe for in vivo diagnosis and in vivo therapy. Herein, we describe the synthetic efforts to prepare conjugates between Sgc8-c and different metallic ions chelator moieties in short times, high purities, and adequate yields. The selected chelator moieties, derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 2-benzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid, and 6-hydrazinonicotinic acid, were covalently attached at the 5'-aptamer position yielding the expected products which were stable in aqueous solution up to 75°C and in typical aptamer storage conditions at least for 30 days. © 2017 John Wiley & Sons A/S.

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xin-xing; Wang, Jian, E-mail: dr_wangjian@yahoo.com.cn; Wang, Hao-lu

2012-03-23

Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substratemore » Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.« less

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

NASA Astrophysics Data System (ADS)

Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

2017-06-01

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Bosak, T.; Pearson, A.; Losick, R.

2005-12-01

Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

NASA Technical Reports Server (NTRS)

Whitson, P. A.; Huls, M. H.; Sams, C. F.

1991-01-01

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

PubMed

Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

2016-03-01

Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively. © 2016 The Protein Society.

Adenylyl cyclase G is activated by an intramolecular osmosensor.

PubMed

Saran, Shweta; Schaap, Pauline

2004-03-01

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

siRNA Targeting of the SNCG Gene Inhibits the Growth of Gastric Carcinoma SGC7901 Cells in vitro and in vivo by Downregulating the Phosphorylation of AKT/ERK.

PubMed

Fan, Changru; Liu, Jinju; Tian, Jianhai; Zhang, Yuliang; Yan, Maojun; Zhu, Chaoyu

2018-06-15

The aim of the study was to evaluate the effects of synuclein-γ (SNCG) silencing on gastric cancer SGC7901 cells and to elucidate the associated mechanisms. pGCSIL-lentiviral siRNA targeting of the SNCG gene was employed to inhibit SNCG expression. Several experiments such as quantitative real-time PCR, Western blotting, MTT, colony formation, migration assay, and flow cytometry were performed to investigate the biological behavior of infected SGC7901 cells. BALB/c nude mice were used as tumor xenograft models to assess the effects of SNCG silencing on tumor growth. Western blot analysis was carried out to determine the relative levels of AKT, p-AKT, ERK, and p-ERK expression. Our results showed that SNCG was overexpressed in SGC7901 cells as compared to normal gastric mucosal epithelial cells. SGC7901 cells transfected with SNCG siRNA demonstrated significantly decreased gastric cancer growth (p < 0.01), reduced cell migration, cell cycle arrest in the G0/G1 phase, promoted tumor cell apoptosis (p < 0.01), and inhibited tumorigenesis in xenograft animal models. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA group than in the control groups. The results of the present study suggest that SNCG siRNA plays a significant role in the proliferation, migration, and tumorigenesis of gastric cancer by downregulating the phosphorylation of AKT and ERK. RNA interference-mediated silencing of SNCG may provide an opportunity to develop a novel treatment strategy for gastric cancer. © 2018 S. Karger AG, Basel.

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.

PubMed

Blain-Hartung, Matthew; Rockwell, Nathan C; Moreno, Marcus V; Martin, Shelley S; Gan, Fei; Bryant, Donald A; Lagarias, J Clark

2018-06-01

Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleus sp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase

PubMed Central

Baum, Paul D.; Sullam, Paul M.; Stoddart, Cheryl A.; McCune, Joseph M.

2011-01-01

Objective To provide a molecular mechanism that explains the association of the antiretroviral guanosine analogue, abacavir, with an increased risk of myocardial infarction. Design Drug effects were studied with biochemical and cellular assays. Methods Human platelets were incubated with nucleoside analogue drugs ex vivo. Platelet activation stimulated by ADP was studied by measuring surface P-selectin with flow cytometry. Inhibition of purified soluble guanylyl cyclase was quantified using an ELISA to measure cGMP production. Results Pre-incubation of platelets in abacavir significantly increased activation in response to ADP in a time and dose-dependent manner. The active anabolite of abacavir, carbovir triphosphate, competitively inhibited soluble guanylyl cyclase activity with a Ki of 55 μmol/l. Conclusion Abacavir competitively inhibits guanylyl cyclase, leading to platelet hyper-reactivity. This may explain the observed increased risk of myocardial infarction in HIV patients taking abacavir. PMID:21941165

Regulation of Mu and Delta Opioid Action in Normal and Morphine-Tolerant Cells and Cell Membrane Preparations

DTIC Science & Technology

1988-03-10

Burns et al., 1975; Aktories et al., 1979), and the lutropin/ choriogonadotropin receptors on porcine luteal membranes (Buettner and Ascoli, 1984...guanyl nucleotide-, fluoride-, and hormone-stimulated adenylyl cyclase activity in the Gs deficient eye- variant of S49 murine lymphoma cells. The...binding was also observed in the as- deficient eye- S49lymphoma cells (Minuth and Jakobs, 1986). Therefore it is highly unlikely that sodium regulates

A flavonoid compound from Chrysosplenium nudicaule inhibits growth and induces apoptosis of the human stomach cancer cell line SGC-7901.

PubMed

Luo, Yanping; Yu, Hongjuan; Yang, Yunshang; Tian, Weihua; Dong, Kaizhong; Shan, Jinyu; Ma, Xingming

2016-07-01

Gastric cancer remains highly prevalent, but treatment options are limited. Natural products have proved to be a rich source of anticancer drugs. Chrysosplenium nudicaule Ledeb. (Saxifragaceae) is a perennial herb that grows in the highlands of China. It has been used as a traditional Chinese medicine to treat digestive diseases for hundreds of years. Recent studies revealed that this herb had anticancer activity, and the flavonoids were speculated to be the effective components. 6,7,3'-Trimethoxy-3,5,4'-trihydroxy flavone (TTF) and 5,4'-dihydroxy-3,6,3'trimethoxy-flavone-7-O-β-d-glucoside (DTFG) are flavonoid compounds isolated from Chrysosplenium nudicaule. This study examined the effect of TTF and DTFG on SGC-7901 human stomach cancer cell in vitro to determine the anticancer and induction of apoptosis properties of TTF. The proliferation of cells treated with 32, 16, 8, 4, and 2 μg/mL of TTF or DTFG for 24, 48, and 72 h was assessed by the MTT assay. After being treated with TTF, the apoptosis of SGC-7901 cells was assessed by acridine orange staining, ultrastructure, electrophoresis of DNA fragmentation, and flow cytometry. Results indicated that TTF inhibited the growth of cancer cells with an IC50 value of 8.33 μg/mL after 72 h incubation. However, DTFG showed no inhibitory effect on the growth of the cancer cell. Further studies on TTF also confirmed that it was able to induce apoptosis of SGC-7901 cells at a concentration as low as 4 μg/mL. The apoptotic effect of TTF makes it a promising candidate for future chemotherapeutic application in treating stomach cancer.

Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

PubMed

Han, P; Lucero, M T

2005-01-01

Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-05-01

Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

Nitric oxide (NO), the only nitrogen monoxide redox form capable of activating soluble guanylyl cyclase.

PubMed

Dierks, E A; Burstyn, J N

1996-06-28

In the present study, we determined that of the redox forms of nitrogen monoxide, NO-, NO and NO+, only NO significantly activates soluble guanylyl cyclase (GTP pyrophosphate-lyase cyclizing, EC 4.6.1.2). Neither of the NO-donors tested, Angeli's salt (Na2N2O3) or Piloty's acid (C6H5SO2NHOH), caused a change in the guanylyl cyclase activity relative to the basal activity level. Interference by other reaction products was eliminated as a possible explanation for the lack of activation. To the extent that NO+ could be stabilized in aqueous solution, by dissolution of the nitrosonium salt NOPF6 in dry organic solvent prior to addition to the enzyme in buffer, NO+ had no effect on the activity of soluble guanylyl cyclase. The counter-ion, PF6-, had a minimal effect on the enzyme activity and, therefore was, not responsible for the lack of activation by NO+. These observations suggest that NO- is the natural activator of soluble guanylyl cyclase and is reasonably identical with endothelium-derived relaxing factor, the physiological regulator of soluble guanylyl cyclase activity.

Ca2+ -dependent regulation of phototransduction.

PubMed

Stephen, Ricardo; Filipek, Sławomir; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2008-01-01

Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca(2+) concentration in the photoreceptor cytosol sensed by Ca(2+)-binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca(2+) concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca(2+)-triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca(2+) sensors in the modulation of phototransduction.

Cyclic GMP-dependent protein kinase-I in the guinea pig cochlea.

PubMed

Tian, F; Fessenden, J D; Schacht, J

1999-05-01

Recent studies have begun to characterize the nitric oxide/cyclic GMP/protein kinase G pathway in the mammalian cochlea by demonstrating the presence of both the enzyme that produces nitric oxide (NO), nitric oxide synthase, and the NO receptor, soluble guanylate cyclase. The present study investigated protein kinase G (cyclic GMP-dependent protein kinase-I, cGK-I), the downstream enzyme of this pathway that frequently mediates its physiological effects. A commercial antibody to a human cGK-I sequence recognized a protein of appropriate molecular weight in Western blots of guinea pig aorta. Immunostaining of guinea pig aorta was consistent with the expected distribution of cGK-I. In lateral wall tissues of the cochlea, pericytes lining the blood vessels of the spiral ligament were strongly immunoreactive. In the organ of Corti, cGK-I was detected in Hensen's, Deiters', and pillar cells, but not in inner and outer hair cells. This distribution coincides with the localization of soluble guanylate cyclase activity and suggests that cGK-I mediates the effects of the NO/cyclic GMP pathway in the cochlea. It reinforces the hypothesis that the NO/cyclic GMP/cGK-I pathway is involved in regulation of cochlear blood flow and supporting cell physiology.

A comparative study of sildenafil, NCX-911 and BAY41-2272 on the anococcygeus muscle of diabetic rats.

PubMed

Kalsi, Jasjit S; Ralph, David J; Madge, David J; Kell, Phil D; Cellek, Selim

2004-12-01

We compared the effects of a nitric oxide (NO)-releasing sildenafil (NCX-911), NO-independent soluble guanylate cyclase activator (BAY41-2272) and sildenafil on the anococcygeus muscle from streptozotocin-induced 16-weeks diabetic rats. NCX-911, BAY41-2272 and sildenafil reduced the phenylephrine-induced tone in the control group (EC50=1088.8+/-165.0, 151.6+/-9.3 and 827.1+/-167.3 nM, respectively). The potencies of NCX-911 and BAY41-2272 were not altered, but that of sildenafil was significantly reduced in the diabetic group. EC50 values for NCX-911, BAY41-2272 and sildenafil in the diabetic group were 1765.9+/-303.5, 209.7+/-27.3 and 2842.2+/-640.3 nM, respectively (P<0.05 for sildenafil). Nitrergic relaxation responses were significantly decreased in the diabetic group. The remaining nitrergic relaxation responses were potentiated by BAY41-2272 but not by sildenafil or NCX-911. These results confirm that endogenous NO derived from nitrergic nerves is significantly decreased in diabetes, and suggest that NO-releasing PDE5 inhibitors and NO-independent soluble guanylate cyclase activators could be more useful than PDE5 inhibitors in the treatment of ED in long-term diabetes.

Effects of nitric oxide on red blood cell deformability.

PubMed

Bor-Kucukatay, Melek; Wenby, Rosalinda B; Meiselman, Herbert J; Baskurt, Oguz K

2003-05-01

In addition to its known action on vascular smooth muscle, nitric oxide (NO) has been suggested to have cardiovascular effects via regulation of red blood cell (RBC) deformability. The present study was designed to further explore this possibility. Human RBCs in autologous plasma were incubated for 1 h with NO synthase (NOS) inhibitors [N(omega)-nitro-l-arginine methyl ester (l-NAME) and S-methylisothiourea], NO donors [sodium nitroprusside (SNP) and diethylenetriamine (DETA)-NONOate], an NO precursor (l-arginine), soluble guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and methylene blue), and a potassium channel blocker [triethylammonium (TEA)]. After incubation, RBC deformability at various shear stresses was determined by ektacytometry. Both NOS inhibitors significantly reduced RBC deformability above a threshold concentration, whereas the NO donors increased deformability at optimal concentrations. NO donors, as well as the NO precursor l-arginine and the potassium blocker TEA, were able to reverse the effects of NOS inhibitors. Guanylate cyclase inhibition reduced RBC deformation, with both SNP and DETA-NONOate able to reverse this effect. These results thus indicate the importance of NO as a determinant of RBC mechanical behavior and suggest its regulatory role for normal RBC deformability.

Effects of Forskolin on Kupffer Cell Production of Interleukin-10 and Tumor Necrosis Factor Alpha Differ from Those of Endogenous Adenylyl Cyclase Activators: Possible Role for Adenylyl Cyclase 9

PubMed Central

Dahle, Maria K.; Myhre, Anders E.; Aasen, Ansgar O.; Wang, Jacob E.

2005-01-01

Proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-α expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and β-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 μg/ml) caused attenuated TNF-α levels in culture medium (forskolin/isoproterenol, P ≤ 0.05; prostaglandin E2, P ≤ 0.01). Forskolin also reduced IL-10 mRNA and protein (P ≤ 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P ≤ 0.05). We conclude that regulation of TNF-α and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2 differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9. PMID:16239525

Cigarette Smoke-Induced Emphysema and Pulmonary Hypertension Can Be Prevented by Phosphodiesterase 4 and 5 Inhibition in Mice

PubMed Central

Pichl, Alexandra; Bednorz, Mariola; Ghofrani, Hossein Ardeschir; Schermuly, Ralph Theo; Seeger, Werner; Grimminger, Friedrich; Weissmann, Norbert

2015-01-01

Rationale Chronic obstructive pulmonary disease (COPD) is a widespread disease, with no curative therapies available. Recent findings suggest a key role of NO and sGC-cGMP signaling for the pathogenesis of the disease. Previous data suggest a downregulation/inactivation of the cGMP producing soluble guanylate cyclase, and sGC stimulation prevented cigarette smoke-induced emphysema and pulmonary hypertension (PH) in mice. We thus aimed to investigate if the inhibition of the cGMP degrading phosphodiesterase (PDE)5 has similar effects. Results were compared to the effects of a PDE 4 inhibitor (cAMP elevating) and a combination of both. Methods C57BL6/J mice were chronically exposed to cigarette smoke and in parallel either treated with Tadalafil (PDE5 inhibitor), Piclamilast (PDE4 inhibitor) or both. Functional measurements (lung compliance, hemodynamics) and structural investigations (alveolar and vascular morphometry) as well as the heart ratio were determined after 6 months of tobacco smoke exposure. In addition, the number of alveolar macrophages in the respective lungs was counted. Results Preventive treatment with Tadalafil, Piclamilast or a combination of both almost completely prevented the development of emphysema, the increase in lung compliance, tidal volume, structural remodeling of the lung vasculature, right ventricular systolic pressure, and right ventricular hypertrophy induced by cigarette smoke exposure. Single, but not combination treatment prevented or reduced smoke-induced increase in alveolar macrophages. Conclusion Cigarette smoke-induced emphysema and PH could be prevented by inhibition of the phosphodiesterases 4 and 5 in mice. PMID:26058042

CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain.

PubMed Central

Ceni, Claire; Pochon, Nathalie; Brun, Virginie; Muller-Steffner, Hélène; Andrieux, Annie; Grunwald, Didier; Schuber, Francis; De Waard, Michel; Lund, Frances; Villaz, Michel; Moutin, Marie-Jo

2003-01-01

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue. PMID

Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

PubMed

Cao, Tian-Jun; Huang, Xing-Qi; Qu, Yuan-Yuan; Zhuang, Zhong; Deng, Yin-Yin; Lu, Shan

2017-04-11

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene ( BfLCYB ) encoding a lycopene β-cyclase from Bangia fuscopurpurea , a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

Effect of ginger, Paullinia cupana, muira puama and l- citrulline, singly or in combination, on modulation of the inducible nitric oxide- NO-cGMP pathway in rat penile smooth muscle cells.

PubMed

Ferrini, Monica G; Garcia, Eduardo; Abraham, Andrea; Artaza, Jorge N; Nguyen, Sabine; Rajfer, Jacob

2018-06-01

COMP-4 is a natural compound-based dietary supplement consisting of the combination of ginger, Paullinia cupana, muira puama and l-citrulline, which when given long-term has been shown in the aged rat to a) upregulate iNOS in the penile smooth muscle cells (SMC), b) reverse the corporal SMC apoptosis and fibrosis associated with corporal veno-occlusive dysfunction (CVOD), and c) improve resulting erectile function. To elucidate the mechanism of how COMP-4 and its individual components modulate the iNOS-cGMP pathway, an in vitro study was conducted using a rat corporal primary SMC culture to determine its effect on NOS, soluble guanylate cyclase (sGC), cGMP and the phosphodiesterase 5 enzyme (PDE5). Primary SMC cultures using the explant technique were initiated by cutting small pieces of corporal tissue from 8 week old Sprague-Dawley rats. The SMC were grown in Dulbecco media with 20% fetal calf serum. The SMC were then incubated with or without COMP-4 (0.69 mg/ml) or its ingredients alone (ginger: 0.225 mg/ml; muira puama, Paullinia cupana and l-citrulline each at 0.9 mg/ml) for up to 24 h mRNA and protein were extracted and used for the determination of NOS, sGC and PDE5 content. cGMP content was determined by ELISA. L-NIL (4 μM) was used as an inhibitor of iNOS activity. Compared to the control values, COMP-4 upregulated expression of cGMP by 85%, induced a 42 fold increase in sGC as well as a 15 fold increase in both iNOS protein and mRNA content while it decreased both PDE5 mRNA and protein content each by about 50%. L-NIL completely inhibited the effect of COMP-4 on cGMP production. When compared with each of the individual four components of COMP-4, it appears that COMP-4 itself had the most profound effect in modulating each one the specific steps within the iNOS-cGMP pathway. This in vitro study demonstrates that COMP-4 is capable of activating the endogenous cellular iNOS-cGMP pathway within the CSM cells, which is theorized to be responsible

Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

PubMed

Chen, S; Lane, A P; Bock, R; Leinders-Zufall, T; Zufall, F

2000-07-01

Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

Small-molecule inhibitors at the PSD-95/nNOS interface protect against glutamate-induced neuronal atrophy in primary cortical neurons.

PubMed

Doucet, M V; O'Toole, E; Connor, T; Harkin, A

2015-08-20

Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/n

The estrogen-dependent baroreflex dysfunction caused by nicotine in female rats is mediated via NOS/HO inhibition: Role of sGC/PI3K/MAPK{sub ERK}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fouda, Mohamed A.; El-Gowelli, Hanan M.; El-Gowilly, Sahar M.

We have previously reported that estrogen (E2) exacerbates the depressant effect of chronic nicotine on arterial baroreceptor activity in female rats. Here, we tested the hypothesis that this nicotine effect is modulated by nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream soluble guanylate cyclase (sGC)/phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinases (MAPKs) signaling. We investigated the effects of (i) inhibition or facilitation of NOS or HO on the interaction of nicotine (2 mg/kg/day i.p., 2 weeks) with reflex bradycardic responses to phenylephrine in ovariectomized (OVX) rats treated with E2 or vehicle, and (ii) central pharmacologic inhibition of sGC, PI3K,more » or MAPKs on the interaction. The data showed that the attenuation by nicotine of reflex bradycardia in OVXE2 rats was abolished after treatment with hemin (HO inducer) or L-arginine (NOS substrate). The hemin or L-arginine effect disappeared after inhibition of NOS (Nω-Nitro-L-arginine methyl ester hydrochloride, L-NAME) and HO (zinc protoporphyrin IX, ZnPP), respectively, denoting the interaction between the two enzymatic pathways. E2-receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but had no effect on baroreflex improvement induced by hemin or L-arginine. Moreover, baroreflex enhancement by hemin was eliminated following intracisternal (i.c.) administration of wortmannin, ODQ, or PD98059 (inhibitors of PI3K, sGC, and extracellular signal-regulated kinases, MAPK{sub ERK}, respectively). In contrast, the hemin effect was preserved after inhibition of MAPK{sub p38} (SB203580) or MAPK{sub JNK} (SP600125). Overall, NOS/HO interruption underlies baroreflex dysfunction caused by nicotine in female rats and the facilitation of NOS/HO-coupled sGC/PI3K/MAPK{sub ERK} signaling might rectify the nicotine effect. - Highlights: • Hemin or L-arginine blunts baroreflex dysfunction caused by nicotine in OVXE2 rats. • NO/CO crosstalk mediates

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

Activated Macrophages Destroy Intracellular Leishmania Major Amastigotes by an l-Arginine-Dependent Killing Mechanism

DTIC Science & Technology

1990-01-01

atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe- dependent enzymatic pathways vital to the survival of amastigotes within...geneti- a precursor of the nitrite measured, may disrupt Fe- cally susceptible BALB/c mice. The exact role of IFN-1 in dependent enzymatic pathways vital...induces the heme - dependent activation of 0 6 ± 4 89 80 guanylate cyclase. with the subsequent stimulation of 0.01 8 ± 3 85 67 the secondary messenger

One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases.

PubMed

Cunningham, F X; Gantt, E

2001-02-27

Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein). Carotenoids with two epsilon-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings. An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

PubMed Central

Cunningham, Francis X.; Gantt, Elisabeth

2001-01-01

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures. PMID:11226339

Primary structure of a guanyl-specific ribonuclease from the fungus Penicillium brevicompactum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulikov, V.A.; Shlyapnikov, S.V.; Yakovlev, G.I.

1986-01-01

By the automatic Edman degradation of the intact S-carboxymethylated protein and a mixture of the products of its proteolytic cleavage at Arg, Lys, and Glu residues, together with results on the kinetics of the proteolysis of the protein under the action of carboxypeptidase Y, the primary structure of the extracellular guanyl-specific RNase of the fungus Penicillium brevicompactum has been determined. The RNase contains 102 amino acid residues: 7 Asp, 7 Asn, 9 Thr, 11 Ser, 4 Glu, 1 Gln, 4 Pro, 10 Gly, 11 Ala, 4 Cys, 7 Val, 4 Ile, 3 Leu, 9 Tyr, 5 Phe, 2 Lys, 3more » His, 1 Arg (M/sub r/ 10,801). It has been established that four hemicystine residues of the P. compactum RNase form, in pairs, two disulfide bonds« less

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

PubMed Central

Rotcheewaphan, Suwatchareeporn; Belisle, John T.; Webb, Kristofor J.; Kim, Hee-Jin; Spencer, John S.

2016-01-01

The second messenger, bis-(3′,5′)-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host–pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography–mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae. PMID:27450520

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

PubMed

Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

2016-09-01

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

The hemodynamic effects of methylene blue when administered at the onset of cardiopulmonary bypass.

PubMed

Maslow, Andrew D; Stearns, Gary; Butala, Parag; Batula, Parag; Schwartz, Carl S; Gough, Jeffrey; Singh, Arun K

2006-07-01

Hypotension occurs during cardiopulmonary bypass (CPB), in part because of induction of the inflammatory response, for which nitric oxide and guanylate cyclase play a central role. In this study we examined the hemodynamic effects of methylene blue (MB), an inhibitor of guanylate cyclase, administered during cardiopulmonary bypass (CPB) to patients taking angiotensin-converting enzyme inhibitors. Thirty patients undergoing cardiac surgery were randomized to receive either MB (3 mg/kg) or saline (S) after institution of CPB and cardioplegic arrest. CPB was managed similarly for all study patients. Hemodynamic data were assessed before, during, and after CPB. The use of vasopressors was recorded. All study patients experienced a similar reduction in mean arterial blood pressure (MAP) and systemic vascular resistance (SVR) with the onset of CPB and cardioplegic arrest. MB increased MAP and SVR and this effect lasted for 40 minutes. The saline group demonstrated a persistently reduced MAP and SVR throughout CPB. The saline group received phenylephrine more frequently during CPB, and more norepinephrine after CPB to maintain a desirable MAP. The MB group recorded significantly lower serum lactate levels despite equal or greater MAP and SVR. In conclusion, administration of MB after institution of CPB for patients taking angiotensin-converting enzyme inhibitors increased MAP and SVR and reduced the need for vasopressors. Furthermore, serum lactate levels were lower in MB patients, suggesting more favorable tissue perfusion.

Nitric oxide-dependent vasorelaxation induced by extractive solutions and fractions of Maytenus ilicifolia Mart ex Reissek (Celastraceae) leaves.

PubMed

Rattmann, Yanna D; Cipriani, Thales R; Sassaki, Guilherme L; Iacomini, Marcello; Rieck, Lia; Marques, Maria C A; da Silva-Santos, José E

2006-04-06

This study reveals that an ethanolic supernatant obtained from an aqueous extractive solution prepared from residues of methanolic extracts of ground leaves of Maytenus ilicifolia is able to cause a concentration- and endothelium-dependent relaxation in pre-contract rat aorta rings, with EC(50) of 199.7 (190-210) microg/ml. The non-selective nitric oxide synthase inhibitors l-NAME and l-NMMA abolished this effect, while superoxide dismutase and MnTBAP (a non-enzymatic superoxide dismutase mimetic) enhanced it. Further, relaxation induced by this ethanolic supernatant have been strongly inhibited by the guanylate cyclase inhibitors methylene blue and ODQ, as well as by the potassium channel blockers 4-aminopyridine and tetraethylammonium, but was unchanged by the cyclooxygenase inhibitor indomethacin and the membrane receptor antagonists atropine, HOE-140 and pirilamine. Partition of the ethanolic supernatant between H(2)O and EtOAc generated a fraction several times more potent, able to fully relax endothelium-intact aorta rings with an EC(50) of 4.3 (3.9-4.8) microg/ml. (13)C NMR spectrum of this fraction showed signals typical of catechin. This study reveals that the leaves of M. ilicifolia possess one or more potent substances able to relax endothelium-intact rat aorta rings, an event that appears to involve nitric oxide production, guanylate cyclase activation and potassium channel opening.

Desensitization of atriopeptin stimulated accumulation and extrusion of cyclic GMP from a kidney epithelial cell line (MDCK).

PubMed

Woods, M; Houslay, M D

1991-02-01

Atriopeptin caused dose- (EC50 ca. 2 x 10(-8) M) and time-dependent increases in the intracellular concentration of cyclic GMP in the MDCK kidney epithelial cell line; an effect potentiated by the phosphodiesterase inhibitor, IBMX. The atriopeptin-catalysed increase in cyclic GMP was transient and reached a maximum some 10-20 min after challenge of cells with atriopeptin. The basis for the transience of this increase was shown to be due to the desensitization of guanylate cyclase coupled with extrusion of cyclic GMP from the cells and the degradation of cyclic GMP by phosphodiesterase activity. Atriopeptin-catalysed extrusion of cyclic GMP was time- and dose-(EC50 ca. 1.5 x 10(-8) M) dependent and was inhibited by probenecid but not by high external cyclic GMP concentrations. The extrusion process underwent apparent desensitization as did guanylate cyclase with similar half lives (T1/2 of ca. 20 min). Desensitization was dose-dependent upon atriopeptin and did not appear to be mediated by elevated cyclic GMP concentrations as pre-incubation with 8-bromo cyclic GMP did not cause desensitization and the half-times for desensitization were similar whether or not IBMX was present. The majority of the cyclic nucleotide phosphodiesterase activity was found in the cytosol fraction of the cells and could be separated into two cyclic AMP specific forms and two cyclic GMP preferring forms.

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

1987-06-25

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

Adenylyl cyclases in the digestive system

PubMed Central

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

2015-01-01

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 Ca2+ 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. PMID:24521753

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.

Nitric oxide, can it be only good? Increasing the antioxidant properties of nitric oxide in hepatocytes by YC-1 compound.

PubMed

Aharoni-Simon, Michal; Anavi, Sarit; Beifuss, Uwe; Madar, Zecharia; Tirosh, Oren

2012-12-01

The aim of the study was to evaluate the effect of Nitric oxide (NO) on redox changes and fat accumulation in hepatocytes. AML-12 hepatocytes were exposed to the NO donor Diethylenetriamine-NONOate (DETA-NO). DETA-NO led to a dose- and time-dependent increase in lipid accumulation in the cells, measured by Nile red fluorescence. Exposure of the cells to 1mM DETA-NO for 24h increased reactive oxygen species production, mainly peroxides. At the same time, NO induced elevation of reduced glutathione (GSH) and a mild activation of the antioxidant transcription factors Hypoxia-inducible factor 1α (HIF1α) and NF-E2 related factor 2 (Nrf-2). We used 100 μM YC-1 to inhibit HIF1α activity and induce activation of soluble Guanylate Cyclase (sGC). YC-1 alone did not affect fat accumulation, and only moderately increased the expression of Nrf-2-targeted genes Heme oxygenase 1 (Hmox1), NAD(P)H dehydrogenase (quinone 1) (Nqo1) and Glutathione S-transferase α1 (Gstα1). However, YC-1 abolished the negative effect of NO on fat accumulation when administered together. Strikingly, YC-1 potentiated the effect of NO on Nrf-2 activation, thus increasing dramatically the antioxidant properties of NO. Moreover, YC-1 intensified the effect of NO on the expression of peroxisome-proliferator-activated receptor-gamma co-activator 1α (PGC1α) and mitochondrial biogenesis markers. This study suggests that YC-1 may shift the deleterious effects of NO into the beneficial ones, and may improve the antioxidant properties of NO. 2012 Elsevier Inc. All rights reserved

A redox-based mechanism for nitric oxide-induced inhibition of DNA synthesis in human vascular smooth muscle cells

PubMed Central

Bundy, Ruth E; Marczin, Nándor; Chester, Adrian H; Yacoub, Magdi

2000-01-01

The current study explored potential redox mechanisms of nitric oxide (NO)-induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells.Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration-dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO.Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO.Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation.Study of extracellular interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release.Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells.These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors. PMID:10742309

Adenosine/nitric oxide crosstalk in the branchial circulation of Squalus acanthias and Anguilla anguilla.

PubMed

Pellegrino, D; Tota, B; Randall, D J

2005-10-01

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

PubMed Central

Barnett, J. E. G.; Rasheed, A.; Corina, D. L.

1973-01-01

After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105. PMID:4352864

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. Copyright © 2014 Elsevier Inc. All rights reserved.

FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms.

PubMed

Mita, Tomohiro; Furuhashi, Masato; Hiramitsu, Shinya; Ishii, Junnichi; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Tanaka, Marenao; Ohno, Kohei; Akasaka, Hiroshi; Ohnishi, Hirofumi; Yoshida, Hideaki; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2015-02-01

Fatty acid-binding protein 4 (FABP4) is expressed in adipocytes, and elevated plasma FABP4 level is associated with obesity-mediated metabolic phenotype. Postprandial regulation and secretory signaling of FABP4 has been investigated. Time courses of FABP4 levels were examined during an oral glucose tolerance test (OGTT; n=53) or a high-fat test meal eating (n=35). Effects of activators and inhibitors of adenyl cyclase (AC)-protein kinase A (PKA) signaling and guanylyl cyclase (GC)-protein kinase G (PKG) signaling on FABP4 secretion from mouse 3T3-L1 adipocytes were investigated. FABP4 level significantly declined after the OGTT or a high-fat meal eating, while insulin level was increased. Treatment with low and high glucose concentration or palmitate for 2 h did not affect FABP4 secretion from 3T3-L1 adipocytes. FABP4 secretion was increased by stimulation of lipolysis using isoproterenol, a β3 -adrenoceptor agonist (CL316243), forskolin, dibutyryl-cAMP and atrial natriuretic peptide, and the induced FABP4 secretion was suppressed by insulin or an inhibitor of PKA (H-89), PKG (KT5823) or hormone sensitive lipase (CAY10499). FABP4 is secreted from adipocytes in association with lipolysis regulated by AC-PKA- and GC-PKG-mediated signal pathways. Plasma FABP4 level declines postprandially, and suppression of FABP4 secretion by insulin-induced anti-lipolytic signaling may be involved in this decline in FABP4 level. © 2014 The Obesity Society.

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.

Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, D.I.; Robbins, J.D.; Ruoho, A.E.

1991-07-15

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolinmore » but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.« less

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

PubMed Central

Ma, H; Gamper, M; Parent, C; Firtel, R A

1997-01-01

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

PubMed Central

Rapiejko, P J; Malbon, C C

1987-01-01

The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate

Corpora lutea in superovulated ewes fed different planes of nutrition.

PubMed

Kraisoon, A; Redmer, D A; Bass, C S; Navanukraw, C; Dorsam, S T; Valkov, V; Reyaz, A; Grazul-Bilska, A T

2018-01-01

The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some luteal functions. FSH is widely used to induce multiple follicle development and superovulation. We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes. Therefore, the aim of this study was to determine if FSH-treatment affects (1) ovulation rate; (2) CL weight; (3) cell proliferation; (4) vascularity; (5) expression of endothelial nitric oxide (eNOS) and soluble guanylate cyclase (sGC) proteins; and (6) luteal and serum progesterone (P4) concentration in control (C), overfed (O), and underfed (U) ewes at the early- and mid-luteal phases. In addition, data generated from this study were compared to data obtained from nonsuperovulated sheep and described by Bass et al. Ewes were categorized by weight and randomly assigned into nutrition groups: C (2.14 Mcal/kg; n = 11), O (2xC; n = 12), and U (0.6xC; n = 11). Nutritional treatment was initiated 60 d prior to day 0 of the estrous cycle. Ewes were injected with FSH on day 13-15 of the first estrous cycle, and blood samples and ovaries were collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was determined, and CL was dissected and weighed. CL was fixed for evaluation of expression of Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC proteins using immunohistochemistry and image analysis. From day 0 until tissue collection, C maintained, O gained, and U lost body weight. The CL number was greater (P < 0.03) in C and O than U. Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P < 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01) cell

Nicotine-induced activation of soluble adenylyl cyclase participates in ion transport regulation in mouse tracheal epithelium.

PubMed

Hollenhorst, Monika I; Lips, Katrin S; Kummer, Wolfgang; Fronius, Martin

2012-11-27

Functional nicotinic acetylcholine receptors (nAChR) have been identified in airway epithelia and their location in the apical and basolateral membrane makes them targets for acetylcholine released from neuronal and non-neuronal sources. One function of nAChR in airway epithelia is their involvement in the regulation of transepithelial ion transport by activation of chloride and potassium channels. However, the mechanisms underlying this nicotine-induced activation of ion transport are not fully elucidated. Thus, the aim of this study was to investigate the involvement of adenylyl cyclases in the nicotine-induced ion current in mouse tracheal epithelium. To evaluate the nicotine-mediated changes of transepithelial ion transport processes electrophysiological Ussing chamber measurements were applied and nicotine-induced ion currents were recorded in the absence and presence of adenylyl cyclase inhibitors. The ion current changes induced by nicotine (100 μM, apical) were not altered in the presence of high doses of atropine (25 μM, apical and basolateral), underlining the involvement of nAChR. Experiments with the transmembrane adenylyl cyclase inhibitor 2'5'-dideoxyadenosine (50 μM, apical and basolateral) and the soluble adenylyl cyclase inhibitor KH7 (10 μM, apical and basolateral) both reduced the nicotine-mediated ion current to a similar extent. Yet, a statistically significant reduction was obtained only in the experiments with KH7. This study indicates that nicotine binding to nAChR in mouse tracheal epithelium activates transepithelial ion transport involving adenylyl cyclase activity. This might be important for novel therapeutic strategies targeting epithelial ion transport mediated by the non-neuronal cholinergic system. Copyright © 2012 Elsevier Inc. All rights reserved.

Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat.

PubMed

Zhang, Ruilan; Wang, Lei; Zhang, Li; Chen, Jieli; Zhu, Zhenping; Zhang, Zhenggang; Chopp, Michael

2003-02-21

We investigated the effects of NO on angiogenesis and the synthesis of vascular endothelial growth factor (VEGF) in a model of focal embolic cerebral ischemia in the rat. Compared with control rats, systemic administration of an NO donor, DETANONOate, to rats 24 hours after stroke significantly enlarged vascular perimeters and increased the number of proliferated cerebral endothelial cells and the numbers of newly generated vessels in the ischemic boundary regions, as evaluated by 3-dimensional laser scanning confocal microscopy. Treatment with DETANONOate significantly increased VEGF levels in the ischemic boundary regions as measured by ELISA. A capillary-like tube formation assay was used to investigate whether DETANONOate increases angiogenesis in ischemic brain via activation of soluble guanylate cyclase. DETANONOate-induced capillary-like tube formation was completely inhibited by a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Blocking VEGF activity by a neutralized antibody against VEGF receptor 2 significantly attenuated DETANONOate-induced capillary-like tube formation. Moreover, systemic administration of a phosphodiesterase type 5 inhibitor (Sildenafil) to rats 24 hours after stroke significantly increased angiogenesis in the ischemic boundary regions. Sildenafil and an analog of cyclic guanosine monophosphate (cGMP) also induced capillary-like tube formation. These findings suggest that exogenous NO enhances angiogenesis in ischemic brain, which is mediated by the NO/cGMP pathway. Furthermore, our data suggest that NO, in part via VEGF, may enhance angiogenesis in ischemic brain.

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niles, L.P.; Hashemi, F.

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 =more » 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.« less

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

Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells.

PubMed

Xia, Quan; Zhao, Yingli; Wang, Jiali; Qiao, Wenhao; Zhang, Dongling; Yin, Hao; Xu, Dujuan; Chen, Feihu

2017-07-01

4-amino-2-trifluoromethyl-phenyl retinate (ATPR) was reported to potentially inhibit proliferation and induce differentiation activity in some tumor cells. In this study, a proteomics approach was used to investigate the possible mechanism by screening the differentially expressed protein profiles of SGC-7901 cells before and after ATPR-treatment in vitro. Peptides digested from the total cellular proteins were analyzed by reverse phase LC-MS/MS followed by a label-free quantification analysis. The SEQUEST search engine was used to identify proteins and bioinformatics resources were used to investigate the involved pathways for the differentially expressed proteins. Thirteen down-regulated proteins were identified in the ATPR-treated group. Bioinformatics analysis showed that the effects of ATPR on 14-3-3ε might potentially involve the PI3K-AKT-FOXO pathway and P27Kip1 expression. Western blot and RT-PCR analysis showed that ATPR could inhibit AKT phosphorylation, up-regulate the expression of FOXO1A and P27Kip1 at both the protein and mRNA levels, and down-regulate the cytoplasmic expression of cyclin E and CDK2. ATPR-induced G0/G1 phase arrest and differentiation can be ablated if the P27kip1 gene is silenced with sequence-specific siRNA or in 14-3-3ε overexpression of SGC-7901 cells. ATPR might cause cell cycle arrest and differentiation in SGC-7901 cells by simultaneously inhibiting the phosphorylation of AKT and down-regulating 14-3-3ε. This change would then enhance the inhibition of cyclin E/CDK2 by up-regulating FOXO1A and P27Kip1. Our findings could be of value for finding new drug targets and for developing more effective differentiation inducer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation and therapeutic targeting of peptide-activated receptor guanylyl cyclases

PubMed Central

Potter, Lincoln R.

2016-01-01

Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP. PMID:21185863

Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

PubMed

Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

2010-07-01

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

The Diurnal Oscillation of MAP Kinase and Adenylyl Cyclase Activities in the Hippocampus Depends on the SCN

PubMed Central

Phan, Trongha; Chan, Guy; Sindreu, Carlos; Eckel-Mahan, Kristin; Storm, Daniel R.

2011-01-01

Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN two days after training for contextual fear memory reduced contextual memory measured two weeks after training indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus. PMID:21775607

Sildenafil Stimulates the Expression of Gaseous Monoxide-Generating Enzymes in Vascular Smooth Muscle Cells via Distinct Signaling Pathways

PubMed Central

Liu, Xiao-ming; Peyton, Kelly J.; Wang, Xinhui; Durante, William

2012-01-01

Sildenafil is a cGMP-specific phosphodiesterase type 5 inhibitor that augments cGMP accumulation following the activation of soluble guanylate cyclase (sGC). In this study, we investigated whether sildenafil promotes the production of the sGC-stimulatory gases, carbon monoxide and nitric oxide, by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and nitric oxide synthase (iNOS) in vascular smooth muscle cells (SMCs). Sildenafil increased HO-1 expression and potentiated cytokine-mediated expression of iNOS and NO synthesis by SMCs. The induction of HO-1 was unaffected by the sGC inhibitor 1H-(1,2,4)oxadiazolo[4,3-α]quinozalin-1-one (ODQ) or the (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindol91,2,3-fg:3′,2′,1′-kl)pyrrolo(3,4-i)benzodiazocine-10-carboxylic acid, methyl ester (KT 5823). However, the sildenafil-mediated increase in HO-1 promoter activity was abolished by mutating the antioxidant responsive elements in the promoter or by overexpressing a dominant-negative mutant of NF-E2-related factor-2 (Nrf2). Furthermore, the induction of HO-1 by sildenafil was accompanied by an increase in reactive oxygen species (ROS) and blocked by N-acetyl-L-cysteine and rotenone. In contrast, the enhancement of cytokine-stimulated NO synthesis by sildenafil was prevented by ODQ and the protein kinase A inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1′-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and duplicated by lipophilic analogues of cGMP. In conclusion, these studies demonstrate that sildenafil stimulates the expression of HO-1 and iNOS via the ROS-Nrf2 and sGC-cGMP pathway, respectively. The ability of sildenafil to block the catabolism of cGMP while stimulating the synthesis of sGC-stimulatory gaseous monoxides through the induction of HO-1 and iNOS provides a potent mechanism by which c

A role for calmodulin-stimulated adenylyl cyclases in cocaine sensitization.

PubMed

DiRocco, Derek P; Scheiner, Zachary S; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R

2009-02-25

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1(-/-) and AC8(-/-)) exhibit Ca(2+)-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca(2+)-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs.

A Role for Calmodulin-Stimulated Adenylyl Cyclases in Cocaine Sensitization

PubMed Central

DiRocco, Derek P.; Scheiner, Zachary S.; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R.

2009-01-01

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that while AC1 and AC8 single knockout mice (AC1−/− and AC8−/−) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knockout (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization following chronic cocaine treatment. Because of the known role for the ERK/MAP kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated extracellular signal-regulated kinase (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase positive (ChAT+) interneurons in DKO mice relative to wild-type (WT) controls. Following acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581, and cAMP response element-binding protein (pCREB) at Ser133 following acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. PMID:19244515

Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

PubMed Central

Hasebe, Masaharu

2016-01-01

The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

Pharmacological evidence for the involvement of the NMDA receptor and nitric oxide pathway in the antidepressant-like effect of lamotrigine in the mouse forced swimming test.

PubMed

Ostadhadi, Sattar; Ahangari, Mohammad; Nikoui, Vahid; Norouzi-Javidan, Abbas; Zolfaghari, Samira; Jazaeri, Farahnaz; Chamanara, Mohsen; Akbarian, Reyhaneh; Dehpour, Ahmad-Reza

2016-08-01

Lamotrigine is an anticonvulsant agent that shows clinical antidepressant properties. The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) synthesis in possible antidepressant-like effect of lamotrigine in forced swimming test (FST) in mice. Intraperitoneal administration of lamotrigine (10mg/kg) decreased the immobility time in the FST (P<0.01) without any effect on locomotor activity in the open-field test (OFT), while higher dose of lamotrigine (30mg/kg) reduced the immobility time in the FST (P<0.001) as well as the number of crossings in the OFT. Pretreatment of animals with NMDA (75mg/kg), l-arginine (750mg/kg, a substrate for nitric oxide synthase [NOS]) or sildenafil (5mg/kg, a phosphodiesterase [PDE] 5 inhibitor) reversed the antidepressant-like effect of lamotrigine (10mg/kg) in the FST. Injection of l-nitroarginine methyl ester (l-NAME, 10mg/kg, a non-specific NOS inhibitor), 7-nitroindazole (30mg/kg, a neuronal NOS inhibitor), methylene blue (20mg/kg, an inhibitor of both NOS and soluble guanylate cyclase [sGC]), or MK-801 (0.05mg/kg), ketamine (1mg/kg), and magnesium sulfate (10mg/kg) as NMDA receptor antagonists in combination with a sub-effective dose of lamotrigine (5mg/kg) diminished the immobility time of animals in the FST compared with either drug alone. None of the drugs produced significant effects on the locomotor activity in the OFT. Based on our findings, it is suggested that the antidepressant-like effect of lamotrigine might mediated through inhibition of either NMDA receptors or NO-cGMP synthesis. Copyright © 2016. Published by Elsevier Masson SAS.

Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp

DOE PAGES

Liu, Kuanqing; Myers, Angela R.; Pisithkul, Tippapha; ...

2015-02-05

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. In this paper, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A surveymore » of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). Finally, we propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.« less

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.

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

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

PubMed

Newby, A C; Chrambach, A

1979-02-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

PubMed Central

Newby, Andrew C.; Chrambach, Andreas

1979-01-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

PubMed

Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2016-03-01

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

PubMed

Mosca, E V; Rousseau, J P; Gulemetova, R; Kinkead, R; Wilson, R J A

2015-02-01

What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

ANF-RGC gene motif 669WTAPELL675 is vital for blood pressure regulation: Biochemical mechanism

PubMed Central

Duda, Teresa; Pertzev, Alexandre; Sharma, Rameshwar K.

2013-01-01

ANF-RGC is the prototype membrane guanylate cyclase, both the receptor and the signal transducer of the hormones ANF and BNP. After binding them at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates production of the second messenger, cyclic GMP. This, in turn, controls the physiological processes of blood pressure, cardiovascular function, and fluid secretion, and others: metabolic syndrome, obesity and apoptosis. What is the biochemical mechanism by which this single molecule controls these diverse processes, explicitly of the blood pressure regulation is the subject of the present study. In line with the concept that the structural modules of ANF-RGC are designed to respond to more than one, yet distinctive signals, the study demonstrates the construction of a novel ANF-RGC-In-gene-669WTAPELL675 mouse model. Through this model, the study establishes that 669WTAPELL675 is a vital ANF signal transducer motif of the guanylate cyclase. Its striking physiological features linked with their biochemistry are that (1) it controls the hormonally-dependent cyclic GMP production in the kidney and the adrenal gland; (3) its deletion causes hypertension, and (3) cardiac hypertrophy; and (4) these mice show higher levels of the plasma aldosterone. For the first time, a mere 7-amino acid encoded motif of the mouse gene has been directly linked with the physiological control of the blood pressure regulation, a detailed biochemistry of this linkage has been established and a model for this linkage has been offered. PMID:23464624

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

PubMed

Kasztan, Małgorzata; Piwkowska, Agnieszka; Kreft, Ewelina; Rogacka, Dorota; Audzeyenka, Irena; Szczepanska-Konkel, Mirosława; Jankowski, Maciej

2016-07-01

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes. Copyright © 2016 the American Physiological Society.

Protein and Signaling Networks in Vertebrate Photoreceptor Cells

PubMed Central

Koch, Karl-Wilhelm; Dell’Orco, Daniele

2015-01-01

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca2+-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments. PMID:26635520

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C

PubMed Central

Müller, Thomas; Rasool, Insha; Heinz-Erian, Peter; Mildenberger, Eva; Hülstrunk, Christian; Müller, Andreas; Michaud, Laurent; Koot, Bart G P; Ballauff, Antje; Vodopiutz, Julia; Rosipal, Stefan; Petersen, Britt-Sabina; Franke, Andre; Fuchs, Irene; Witt, Heiko; Zoller, Heinz; Janecke, Andreas R; Visweswariah, Sandhya S

2016-01-01

Objective Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. Design We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. Results We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. Conclusions Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD. PMID:25994218

Ca2+-sensors and ROS-GC: interlocked sensory transduction elements: a review

PubMed Central

Sharma, Rameshwar K.; Duda, Teresa

2012-01-01

From its initial discovery that ROS-GC membrane guanylate cyclase is a mono-modal Ca2+-transduction system linked exclusively with the photo-transduction machinery to the successive finding that it embodies a remarkable bimodal Ca2+ signaling device, its widened transduction role in the general signaling mechanisms of the sensory neuron cells was envisioned. A theoretical concept was proposed where Ca2+-modulates ROS-GC through its generated cyclic GMP via a nearby cyclic nucleotide gated channel and creates a hyper- or depolarized sate in the neuron membrane (Ca2+ Binding Proteins 1:1, 7–11, 2006). The generated electric potential then becomes a mode of transmission of the parent [Ca2+]i signal. Ca2+ and ROS-GC are interlocked messengers in multiple sensory transduction mechanisms. This comprehensive review discusses the developmental stages to the present status of this concept and demonstrates how neuronal Ca2+-sensor (NCS) proteins are the interconnected elements of this elegant ROS-GC transduction system. The focus is on the dynamism of the structural composition of this system, and how it accommodates selectivity and elasticity for the Ca2+ signals to perform multiple tasks linked with the SENSES of vision, smell, and possibly of taste and the pineal gland. An intriguing illustration is provided for the Ca2+ sensor GCAP1 which displays its remarkable ability for its flexibility in function from being a photoreceptor sensor to an odorant receptor sensor. In doing so it reverses its function from an inhibitor of ROS-GC to the stimulator of ONE-GC membrane guanylate cyclase. PMID:22509149

cGMP may have trophic effects on beta cell function comparable to those of cAMP, implying a role for high-dose biotin in prevention/treatment of diabetes.

PubMed

McCarty, Mark F

2006-01-01

Incretin hormones have trophic effects on beta cell function that can aid prevention and treatment of diabetes. cAMP is the primary mediator of these effects, and has been shown to potentiate glucose-stimulated insulin secretion, promote proper beta cells differentiation by increasing expression of the crucial transcription factor PDX-1, and prevent beta cell apoptosis. cGMP's role in beta cell function has received far less scrutiny, but there is emerging evidence that it may have a trophic impact on beta cell function analogous to that of cAMP. An increase in plasma glucose boosts beta cell production of cGMP, which acts as a feed-forward mediator to enhance glucose-stimulated insulin secretion. cGMP also has an anti-apoptotic effect in beta cells, and there is now indirect evidence that it promotes expression of PDX-1. Supraphysiological concentrations of biotin can directly activate guanylate cyclase, and there is limited evidence that high intakes of this vitamin can be therapeutically beneficial in diabetics and in rodent models of diabetes. Beneficial effects of cGMP on muscle insulin sensitivity and on control of hepatic glucose output may contribute to biotin's utility in diabetes. The fact that nitric oxide/cGMP exert a range of favorable effects on vascular health should further encourage exploration of biotin's preventive and therapeutic potential. If an appropriate high-dose biotin regimen could achieve a modest systemic increase in guanylate cyclase activity, without entailing unacceptable side effects or risks, such a regimen might have considerable potential for promoting vascular health and preventing or managing diabetes.

Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration

PubMed Central

Ma, Hongwei; Thapa, Arjun; Morris, Lynsie; Redmond, T. Michael; Baehr, Wolfgang; Ding, Xi-Qin

2014-01-01

Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling, which regulates cell proliferation, differentiation, and apoptosis, plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models. Retinol isomerase RPE65-deficient mice [a model of Leber congenital amaurosis (LCA) with rapid cone loss] and cone photoreceptor function loss type 1 mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone death. Further, cone cyclic nucleotide-gated channel B subunit-deficient mice (moderate achromatopsia) and guanylate cyclase 2e-deficient mice (LCA with slower cone loss) were used to determine whether triiodothyronine (T3) treatment (stimulating TH signaling) causes deterioration of cones. We found that cone density in retinol isomerase RPE65-deficient and cone photoreceptor function loss type 1 mice increased about sixfold following antithyroid treatment. Cone density in cone cyclic nucleotide-gated channel B subunit-deficient and guanylate cyclase 2e-deficient mice decreased about 40% following T3 treatment. The effect of TH signaling on cone viability appears to be independent of its regulation on cone opsin expression. This work demonstrates that suppressing TH signaling in retina dystrophy mouse models is protective of cones, providing insights into cone preservation and therapeutic interventions. PMID:24550448

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.

Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD(+) glycohydrolases.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Schuber, F

2000-01-01

Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism. PMID:10861229

GUCY2D Cone-Rod Dystrophy-6 Is a "Phototransduction Disease" Triggered by Abnormal Calcium Feedback on Retinal Membrane Guanylyl Cyclase 1.

PubMed

Sato, Shinya; Peshenko, Igor V; Olshevskaya, Elena V; Kefalov, Vladimir J; Dizhoor, Alexander M

2018-03-21

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S + rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca 2+ phototransduction feedback and abnormally high free-Ca 2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2 +/+ or GCAP1,2 +/- backgrounds, was prevented in GCAP1,2 -/- mice lacking Ca 2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca 2+ levels in photoreceptors. SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D ), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

PubMed

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

PubMed Central

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

Nitric oxide signaling pathway regulates potassium chloride cotransporter-1 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, M; Lauf, P K; Adragna, N C

2001-11-30

Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.

Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

1986-02-01

This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of suchmore » agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells.« less

Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, G.P.

1987-01-01

Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted inmore » a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.« less

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

DOE PAGES

Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.; ...

2017-01-17

The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identifiedmore » two genes ( qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.« less

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.

The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identifiedmore » two genes ( qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.« less

In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli.

PubMed

Chatterjee, A; Bhattacharya, A K

1988-06-01

The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

PubMed

Gao, Yuansheng; Chen, Zhengju; Leung, Susan W S; Vanhoutte, Paul M

2015-06-01

In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

PubMed Central

Nathanson, James A.; Greengard, Paul

1974-01-01

An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

Functional Lycopene Cyclase (CruA) in Cyanobacterium, Arthrospira platensis NIES-39, and its Role in Carotenoid Synthesis.

PubMed

Sugiyama, Kenjiro; Ebisawa, Masashi; Yamada, Masaharu; Nagashima, Yoshiki; Suzuki, Hideyuki; Maoka, Takashi; Takaichi, Shinichi

2017-04-01

The genus Arthrospira is filamentous, non-nitrogen-fixing cyanobacteria that is commercially important. We identified the molecular structures of carotenoids in Arthrospira platensis NIES-39. The major carotenoid identified was β-carotene. In addition, the hydroxyl derivatives of β-cryptoxanthin and (3R,3'R)-zeaxanthin were also found to be present. The carotenoid glycosides were identified as (3R,2'S)-myxol 2'-methylpentoside and oscillol 2,2'-dimethylpentoside. The methylpentoside moiety was a mixture of fucoside and chinovoside in an approximate ratio of 1 : 4. Trace amounts of the ketocarotenoid 3'-hydroxyechinenone were also found. Three types of lycopene cyclases have been functionally confirmed in carotenogenesis organisms. In cyanobacteria, the functional lycopene cyclases (CrtL, CruA and CruP) have only been found in four species. In this study, we found that CruA exhibited lycopene cyclase activity in transformed Escherichia coli, which contains lycopene, but CruP exhibited no lycopene cyclase activity and crtL was absent. This is the third cyanobacterial species in which CruA activity has been confirmed. Neurosporene was not a substrate of CruA in E. coli, whereas lycopene cyclases of CrtY (bacteria), CrtL (plants) and CrtYB (fungi) have been reported to convert neurosporene to 7,8-dihydro-β-carotene. β-Carotene hydroxylase (CrtR) was found to convert β-carotene to zeaxanthin in transformed E. coli, which contains β-carotene. Among the β-carotene hydroxylases, bacterial CrtZ and eukaryotic CrtR and BCH have similarities, whereas cyanobacterial CrtR appears to belong to another clade. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the A. platensis NIES-39 genome, we propose a biosynthetic pathway for the carotenoids as well as the corresponding genes and enzymes. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved

Renal Integrin-Linked Kinase Depletion Induces Kidney cGMP-Axis Upregulation: Consequences on Basal and Acutely Damaged Renal Function

PubMed Central

Cano-Peñalver, José Luis; Griera, Mercedes; García-Jerez, Andrea; Hatem-Vaquero, Marco; Ruiz-Torres, María Piedad; Rodríguez-Puyol, Diego; de Frutos, Sergio; Rodríguez-Puyol, Manuel

2015-01-01

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis–related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage. PMID:26562149

Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning.

PubMed

Sindreu, Carlos Balet; Scheiner, Zachary S; Storm, Daniel R

2007-01-04

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

PubMed Central

Cynis, Holger; Hoffmann, Torsten; Friedrich, Daniel; Kehlen, Astrid; Gans, Kathrin; Kleinschmidt, Martin; Rahfeld, Jens-Ulrich; Wolf, Raik; Wermann, Michael; Stephan, Anett; Haegele, Monique; Sedlmeier, Reinhard; Graubner, Sigrid; Jagla, Wolfgang; Müller, Anke; Eichentopf, Rico; Heiser, Ulrich; Seifert, Franziska; Quax, Paul H A; de Vries, Margreet R; Hesse, Isabel; Trautwein, Daniela; Wollert, Ulrich; Berg, Sabine; Freyse, Ernst-Joachim; Schilling, Stephan; Demuth, Hans-Ulrich

2011-01-01

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE1-CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis. PMID:21774078

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie

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 tomore » 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.« less

Localization of the action of cholera toxin on adenyl cyclase in mucosal epithelial cells of rabbit intestine

PubMed Central

Parkinson, David K.; Ebel, Hans; DiBona, Donald R.; Sharp, Geoffrey W. G.

1972-01-01

Brush borders and plasma membranes have been purified from mucosal epithelial cells of rabbit ileum under control conditions and after treatment for 3 hr with cholera toxin in vivo. The activity of several enzymes in these preparations was measured. It was concluded that adenyl cyclase, like NaK-ATPase, seems not to be a normal constituent of brush borders. Both these enzymes are present in plasma membrane preparations derived largely from the basal and lateral margins of the epithelial cells, both may be phospholipid dependent enzymes and both are affected by cholera toxin. Adenyl cyclase activity is increased while NaK-ATPase is decreased. The activities of alkaline phosphatase, leucineaminopeptidase, 5′-nucleotidase, glucose-6-phosphatase, and Mg-ATPase were not found to be affected by the toxin. Cholera toxin, which makes contact with the luminal side of the epithelial cells, in the natural disease and in the experimental model, would appear to exert its pathologic effect on adenyl cyclase at the opposite (basal and lateral) side of the cells. Images PMID:4344729

NO, nitrotyrosine, and cyclic GMP in signal transduction

NASA Technical Reports Server (NTRS)

Hanafy, K. A.; Krumenacker, J. S.; Murad, F.

2001-01-01

Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells.

PubMed

Li, Xiaotong; Xie, Hongqing; Chen, Yajie; Lang, Mingzi; Chen, Yuyin; Shi, Liangen

2018-03-28

Silkworm pupae ( Bombyx mori ) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.

The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery.

PubMed

Bradley, Anthony R; Echalier, Aude; Fairhead, Michael; Strain-Damerell, Claire; Brennan, Paul; Bullock, Alex N; Burgess-Brown, Nicola A; Carpenter, Elisabeth P; Gileadi, Opher; Marsden, Brian D; Lee, Wen Hwa; Yue, Wyatt; Bountra, Chas; von Delft, Frank

2017-11-08

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets' genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target's TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets. © 2017 The

DIFFERENT CONCENTRATIONS OF SIJUNZI DECOCTION INHIBIT PROLIFERATION AND INDUCE APOPTOSIS OF HUMAN GASTRIC CANCER SGC-7901 SIDE POPULATION.

PubMed

Qian, Jun; Li, Jing; Jia, Jianguang; Jin, Xin; Yu, Dajun; Guo, Chenxu; Xie, Bo; Qian, Liyu

2016-01-01

Sijunzi Decoction (SD) is a traditional Chinese medicine which is composed of Ginseng, Atractylodes, Poria and Licorice. It is one of the commonly used Chinese traditional medicines that showed anti-gastric cancer activity in clinical studies. Previous evidence demonstrated SD parties (Ginseng, Atractylodes, Poria, Licorice) can inhibit proliferation and induced apoptosis for gastric cancer cell. In order to further investigate the anticancer effect of SD in gastric cancer, we observed the effects of different concentrations of SD on proliferation and apoptosis of Side Population Cells (SP) of human gastric cancer SGC-7901. SGC-7901 SP and Non- Side Population Cells (NSP) were sorted through flow cytometry; to detect the changes of proliferation of SP and NSP before and after the intervention of serum containing different concentrations of SD using cck-8 method; to detect the changes of cell cycle and apoptosis of SP and NSP before and after the intervention of serum containing different concentrations of SD through flow cytometry; to detect the effects of serum containing different concentrations of SD on apoptosis-related proteins Bax and Bcl-2 of SP and NSP before and after the intervention by western-blot. It was found that different concentrations of SD serum treatments inhibited cell proliferation in a time-dependent and concentration-dependent manner. Compared with the control group (normal saline serum treatment), there were increase in G1/G0 phase population of SP and NSP, and decrease in G2/M and S phase population ( P <0.05). Meanwhile, we found G1/G0 arrest induced by different concentrations of SD serum which was followed by apoptosis in a time-dependent and concentration-dependent manner. The apoptosis rate of SD serum treatment group was higher than the control group ( P <0.05), the apoptosis rate of 48 h treatment was higher than 24 h treatment ( P <0.05), and as the SD serum concentration increases, apoptosis rate is higher and higher ( P <0

The development of Sustainability Graduate Community (SGC) as a learning pathway for sustainability education - a framework for engineering programmes in Malaysia Technical Universities Network (MTUN)

NASA Astrophysics Data System (ADS)

Johan, Kartina; Mohd Turan, Faiz

2016-11-01

‘Environmental and sustainability’ is one of the Program Outcome (PO) designated by the Board of Engineers Malaysia (BEM) as one of the accreditation program requirement. However, to-date the implementation of sustainability elements in engineering programme in the technical universities in Malaysia is within individual faculty's curriculum plan and lack of university-level structured learning pathway, which enable all students to have access to an education in sustainability across all disciplines. Sustainability Graduate Community (SGC) is a framework designed to provide a learning pathway in the curriculum of engineering programs to inculcate sustainability education among engineering graduates. This paper aims to study the required attributes in Sustainability Graduate Community (SGC) framework to produce graduates who are not just engineers but also skilful in sustainability competencies using Global Project Management (GPM) P5 Standard for Sustainability. The development of the conceptual framework is to provide a constructive teaching and learning plan for educators and policy makers to work on together in developing the Sustainability Graduates (SG), the new kind of graduates from Malaysia Technical Universities Network (MTUN) in Malaysia who are literate in sustainability practices. The framework also support the call for developing holistic students based on Malaysian Education Blueprint (Higher Education) and address the gap between the statuses of engineering qualification to the expected competencies from industries in Malaysia in particular by achieving the SG attributes outlined in the framework

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

PubMed

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

2014-04-11

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 Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.

Ca2+-Stimulated Adenylyl Cyclases Regulate ERK-Dependent Activation of MSK1 During Fear Conditioning

PubMed Central

Sindreu, Carlos Balet; Scheiner, Zachary S.; Storm, Daniel R.

2007-01-01

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation have not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are co-activated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory. PMID:17196532

H{sub 2}S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Sen; Ping, Na-na; Cao, Lei, E-mail: leicao@mail.xjtu.edu.cn

2015-12-15

Hydrogen sulfide (H{sub 2}S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H{sub 2}S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H{sub 2}S, 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-typemore » Ca{sup 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, H{sub 2}S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. - Highlights: • The vasoactivity effect of NaHS, a donor of H{sub 2}S, was studied on rat cerebral arteries. • H{sub 2}S induces a constriction, not a relaxant effect on basilar arteries. • The vasoconstrictive effect is invovled in inhibiting adenylyl cyclase to reduce cAMP levels. • The vasoconstriction is partially antagonized by NO, and does not necessarily act via NO pathway.« less

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin1[OPEN

PubMed Central

Almeida, Aldo; Khakimov, Bekzod; Bassard, Jean-Etienne; Appendino, Giovanni

2018-01-01

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana. In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism. PMID:29203557

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

PubMed Central

Ostolaza, Helena; Martín, César; González-Bullón, David; Uribe, Kepa B.; Etxaniz, Asier

2017-01-01

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered. PMID:28934133

Acute Hemodynamic Effects of Riociguat in Patients With Pulmonary Hypertension Associated With Diastolic Heart Failure (DILATE-1)

PubMed Central

Pretsch, Ingrid; Steringer-Mascherbauer, Regina; Jansa, Pavel; Rosenkranz, Stephan; Tufaro, Caroline; Bojic, Andja; Lam, Carolyn S. P.; Frey, Reiner; Ochan Kilama, Michael; Unger, Sigrun; Roessig, Lothar; Lang, Irene M.

2014-01-01

BACKGROUND: Deficient nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate signaling results from endothelial dysfunction and may underlie impaired cardiac relaxation in patients with heart failure with preserved left ventricular ejection fraction (HFpEF) and pulmonary hypertension (PH). The acute hemodynamic effects of riociguat, a novel soluble guanylate cyclase stimulator, were characterized in patients with PH and HFpEF. METHODS: Clinically stable patients receiving standard HF therapy with a left ventricular ejection fraction > 50%, mean pulmonary artery pressure (mPAP) ≥ 25 mm Hg, and pulmonary arterial wedge pressure (PAWP) > 15 mm Hg at rest were randomized to single oral doses of placebo or riociguat (0.5, 1, or 2 mg). The primary efficacy variable was the peak decrease in mPAP from baseline up to 6 h. Secondary outcomes included hemodynamic and echocardiographic parameters, safety, and pharmacokinetics. RESULTS: There was no significant change in peak decrease in mPAP with riociguat 2 mg (n = 10) vs placebo (n = 11, P = .6). However, riociguat 2 mg significantly increased stroke volume (+9 mL [95% CI, 0.4-17]; P = .04) and decreased systolic BP (−12 mm Hg [95% CI, −22 to −1]; P = .03) and right ventricular end-diastolic area (−5.6 cm2 [95% CI, −11 to −0.3]; P = .04), without significantly changing heart rate, PAWP, transpulmonary pressure gradient, or pulmonary vascular resistance. Riociguat was well tolerated. CONCLUSIONS: In patients with HFpEF and PH, riociguat was well tolerated, had no significant effect on mPAP, and improved exploratory hemodynamic and echocardiographic parameters. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01172756; URL: www.clinicaltrials.gov PMID:24991733

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change1[W][OA

PubMed Central

Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

2010-01-01

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate. PMID:20460582

P2Y12 shRNA treatment decreases SGC activation to relieve diabetic neuropathic pain in type 2 diabetes mellitus rats.

PubMed

Wang, Shouyu; Wang, Zilin; Li, Lin; Zou, Lifang; Gong, Yingxin; Jia, Tianyu; Zhao, Shanhong; Yuan, Huilong; Shi, Liran; Liu, Shuangmei; Wu, Bing; Yi, Zhihua; Liu, Hui; Gao, Yun; Li, Guilin; Deussing, Jan M; Li, Man; Zhang, Chunping; Liang, Shangdong

2018-06-26

Diabetic neuropathic pain is a common complication of type 2 diabetes mellitus (DM). Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in neuropathic pain through the release of proinflammatory cytokines. The P2Y12 receptor is expressed in SGCs of the DRG. In this study, our aim was to investigate the role of the P2Y12 receptor on the pathological changes in diabetic neuropathic pain. The present study showed that diabetic neuropathic pain increased mechanical and thermal hyperalgesia in type 2 DM model rats. The results showed that the expression levels of P2Y12 messenger RNA (mRNA) and protein in DRG SGCs were increased in DM model rats compared with control rats. Glial fibrillary acidic protein (GFAP) and interleukin-1β (IL-1β) expression levels in the DRG were increased in DM rats. Upregulation of GFAP is a marker of SGC activation. Targeting the P2Y12 receptor by short hairpin RNA (shRNA) decreased the upregulated expression of P2Y12 mRNA and protein, coexpression of P2Y12 and GFAP, the expression of GFAP, IL-1β, and tumor necrosis factor-receptor 1 in the DRG of DM rats, and relieved mechanical and thermal hyperalgesia in DM rats. After treatment with the P2Y12 receptor shRNA, the enhancing integrated OPTICAL density (IOD) ratios of p-P38 MAPK to P38 mitogen activated protein kinase (MAPK) in the DM rats treated with P2Y12 shRNA were significantly lower than that in the untreated DM rats. Therefore, P2Y12 shRNA treatment decreased SGC activation to relieve mechanical and thermal hyperalgesia in DM rats. © 2018 Wiley Periodicals, Inc.

[Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production].

PubMed

Jin, Yingfu; Han, Li; Zhang, Shasha; Li, Shizhong; Liu, Weifeng; Tao, Yong

2017-11-25

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells.

PubMed

Wachten, Sebastian; Masada, Nanako; Ayling, Laura-Jo; Ciruela, Antonio; Nikolaev, Viacheslav O; Lohse, Martin J; Cooper, Dermot M F

2010-01-01

Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.

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

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.

Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conner, D.A.

1988-01-01

Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, whichmore » modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.« less

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.

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa

PubMed Central

Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2015-01-01

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC. PMID:26625288

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

PubMed

Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2015-12-01

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

Influence of volatile anesthetics on muscarinic receptor adenylate cyclase coupling in brain and heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anthony, B.L.

In the present study, the influence of four volatile anesthetics (enflurane, isoflurane, diethyl ether, and chloroform) on (1) muscarinic receptor binding parameters and (2) muscarnic regulation of adenylate cyclase activity was examined using membranes isolated from rat brain and heart. Membranes were equilibrated with each of the four anesthetics for 30 minutes and then during the binding assay. The data obtained can be summarized as follows: (1) volatile anesthetics increased receptor affinity for a radiolabeled antagonists, ({sup 3}H)N-methylscopolamine (({sup 3}H)MS), by decreasing its rate of dissociation in brain stem, but not in cardiac, membranes, (2) volatile anesthetics decreased high affinitymore » ({sup 3}H)Oxotremorine-M binding, (3) volatile anesthetics depressed or eliminated the guanine nucleotide sensitivity of agonist binding. The influence of volatile anesthetics on muscarinic regulation of adenylate cyclase enzyme activity was studied using {alpha}({sup 32}P)ATP as the substrate.« less

The diurnal oscillation of MAP (mitogen-activated protein) kinase and adenylyl cyclase activities in the hippocampus depends on the suprachiasmatic nucleus.

PubMed

Phan, Trongha X; Phan, Trongha H; Chan, Guy C-K; Sindreu, Carlos B; Eckel-Mahan, Kristin L; Storm, Daniel R

2011-07-20

Consolidation of hippocampus-dependent memory is dependent on activation of the cAMP/Erk/MAPK (mitogen-activated protein kinase) signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN 2 d after training for contextual fear memory reduced contextual memory measured 2 weeks after training, indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus.

Ca2+ Modulation of ANF-RGC: New Signaling Paradigm Interlocked with Blood Pressure Regulation

PubMed Central

Duda, Teresa; Pertzev, Alexandre; Sharma, Rameshwar K.

2012-01-01

ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single trans-membrane protein. After binding these hormones at the extracellular domain, ANF-RGC at its intracellular domain signals the activation of the C-terminal catalytic module and accelerates the production of the second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. At present this is the sole transduction mechanism and the physiological function of ANF-RGC. Through comprehensive studies involving biochemistry, immunohistochemistry, and blood pressure measurements in mice with targeted gene deletions, the present study demonstrates a new signaling model of ANF-RGC that also controls blood pressure. In this model (1) ANF-RGC is not the transducer of ANF and BNP; (2) its extracellular domain is not used for signaling; and (3) the signal-flow is not downstream from the extracellular domain to the core catalytic domain. Instead, the signal is the intracellular Ca2+, which is translated at the site of its reception, at the core catalytic domain of ANF-RGC. A model for this Ca2+ signal transduction is diagrammed. It captures Ca2+ through its Ca2+ sensor myristoylated neurocalcin δ and up-regulates ANF-RGC activity with a K1/2 of 0.5 μM. The neurocalcin δ-modulated domain resides in the 849DIVGFTALSAESTPMQVV866 segment of ANF-RGC, which is a part of the core catalytic domain. Thereby, ANF-RGC is primed to receive, transmit and translate the Ca2+ signals into the generation of cyclic GMP at a rapid rate. The study defines a new paradigm of the membrane guanylate cyclase signaling, which is linked to the physiology of cardiac vasculature regulation and possibly also to fluid secretion. PMID:23088492

Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.

PubMed

Kollmann, Heiko; Becker, Simon F; Shirdel, Javid; Scholten, Alexander; Ostendorp, Anna; Lienau, Christoph; Koch, Karl-Wilhelm

2012-06-15

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

The Crystal Structure of GCAP3 Suggests Molecular Mechanism of GCAP–linked Cone Dystrophies

PubMed Central

Stephen, Ricardo; Palczewski, Krzysztof; Sousa, Marcelo C.

2014-01-01

Summary Absorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic–GMP (cGMP). This hydrolysis promotes the closing of cGMP–gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate Cyclase Activating Proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca2+–dependent manner. At high [Ca2+], typical of the dark–adapted state (~500 nM), GCAPs inhibit retinal GCs. At the low [Ca2+] (~50 nM) that occur after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark–state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca2+ bound. GCAP3 is an EF–hand Ca2+–binding protein with Ca2+ bound to EF2, 3 and 4, while Ca2+ binding to EF–hand 1 is disabled. GCAP3 contains two domains with the EF–hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca2+–binding proteins. Residues not involved in Ca2+ binding, but conserved in all GCAPs, cluster around EF1 in the N–terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations. PMID:16626734

The crystal structure of GCAP3 suggests molecular mechanism of GCAP-linked cone dystrophies.

PubMed

Stephen, Ricardo; Palczewski, Krzysztof; Sousa, Marcelo C

2006-06-02

Absorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic-GMP (cGMP). This hydrolysis promotes the closing of cGMP-gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate cyclase-activating proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca2+-dependent manner. At high [Ca2+], typical of the dark-adapted state (approximately 500 nM), GCAPs inhibit retinal GCs. At the low [Ca2+] (approximately 50 nM) that occurs after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark-state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca2+ bound. GCAP3 is an EF-hand Ca2+-binding protein with Ca2+ bound to EF2, 3 and 4, while Ca2+ binding to EF-hand 1 is disabled. GCAP3 contains two domains with the EF-hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca2+-binding proteins. Residues not involved in Ca2+ binding, but conserved in all GCAPs, cluster around EF1 in the N-terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations.

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide andnitroxyl ion

PubMed Central

Wanstall, Janet C; Jeffery, Trina K; Gambino, Agatha; Lovren, Fina; Triggle, Christopher R

2001-01-01

Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO·; NO gas solution) and nitroxyl ion (NO−; from Angeli's salt). The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 μM), concentration-dependently inhibited responses to all agents. 10 μM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. The NO· scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 μM) and hydroxocobalamin (100 μM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. The NO− inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO· and NO−. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO. PMID:11588100

Attenuated response of L-type calcium current to nitric oxide in atrial fibrillation.

PubMed

Rozmaritsa, Nadiia; Christ, Torsten; Van Wagoner, David R; Haase, Hannelore; Stasch, Johannes-Peter; Matschke, Klaus; Ravens, Ursula

2014-03-01

Nitric oxide (NO) synthesized by cardiomyocytes plays an important role in the regulation of cardiac function. Here, we studied the impact of NO signalling on calcium influx in human right atrial myocytes and its relation to atrial fibrillation (AF). Right atrial appendages (RAAs) were obtained from patients in sinus rhythm (SR) and AF. The biotin-switch technique was used to evaluate endogenous S-nitrosylation of the α1C subunit of L-type calcium channels. Comparing SR to AF, S-nitrosylation of Ca(2+) channels was similar. Direct effects of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on L-type calcium current (ICa,L) were studied in cardiomyocytes with standard voltage-clamp techniques. In SR, ICa,L increased with SNAP (100 µM) by 48%, n/N = 117/56, P < 0.001. The SNAP effect on ICa,L involved activation of soluble guanylate cyclase and protein kinase A. Specific inhibition of phosphodiesterase (PDE)3 with cilostamide (1 µM) enhanced ICa,L to a similar extent as SNAP. However, when cAMP was elevated by PDE3 inhibition or β-adrenoceptor stimulation, SNAP reduced ICa,L, pointing to cGMP-cAMP cross-regulation. In AF, the stimulatory effect of SNAP on ICa,L was attenuated, while its inhibitory effect on isoprenaline- or cilostamide-stimulated current was preserved. cGMP elevation with SNAP was comparable between the SR and AF group. Moreover, the expression of PDE3 and soluble guanylate cyclase was not reduced in AF. NO exerts dual effects on ICa,L in SR with an increase of basal and inhibition of cAMP-stimulated current, and in AF NO inhibits only stimulated ICa,L. We conclude that in AF, cGMP regulation of PDE2 is preserved, but regulation of PDE3 is lost.

Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

PubMed

Chetham, P M; Sefton, W D; Bridges, J P; Stevens, T; McMurtry, I F

1997-04-01

Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.

Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

PubMed

Quinn, Sierra N; Graves, Sarai H; Dains-McGahee, Clayton; Friedman, Emilee M; Hassan, Humma; Witkowski, Piotr; Sabbatini, Maria E

2017-04-01

Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1988-01-01

The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

PubMed

Hoang, Van-Hai; Tran, Phuong-Thao; Cui, Minghua; Ngo, Van T H; Ann, Jihyae; Park, Jongmi; Lee, Jiyoun; Choi, Kwanghyun; Cho, Hanyang; Kim, Hee; Ha, Hee-Jin; Hong, Hyun-Seok; Choi, Sun; Kim, Young-Ho; Lee, Jeewoo

2017-03-23

Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of β-amyloid peptides (pGlu-Aβ) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aβ 3E-42 . An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aβ and total Aβ and restored cognitive functions. This potent Aβ-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

Developing grasshopper neurons show variable levels of guanylyl cyclase activity on arrival at their targets.

PubMed

Ball, E E; Truman, J W

1998-04-27

The ability of certain grasshopper neurons to respond to exogenously applied donors of nitric oxide (NO) by producing cyclic GMP (cGMP) depends on their developmental state. ODQ, a selective blocker of NO-sensitive guanylyl cyclase, blocks cGMP production at 10(-5) M, thus confirming the nature of the response. Experiments in which the distal axon is separated from its proximal stump before application of an NO donor show that guanylyl cyclase is distributed uniformly throughout the neuron. In the locust abdomen, where segments are formed sequentially, the pattern of guanylyl cyclase up-regulation is predictable and sequential from anterior to posterior. There are two patterns of innervation by cGMP-expressing motor neurons. In the first, typified by muscle 187, an innervating neuron begins to be NO responsive on arrival at its muscle and continues to be so over most of the remainder of embryonic development, including the formation of motor end plates. In the second, typified by a neuron innervating muscle 191, the neuron extends well along the muscle, apparently laying down a number of sites of contact with it, before it becomes NO responsive. In both patterns, however, NO responsiveness marks the neuron's transition from growth cone elongation to the production of lateral branches. Individual muscles receive innervation from multiple motor neurons, some of which express transient NO sensitivity during development and others which do not. With the exception of the leg motor neuron SETi, the first motor neuron to reach any muscle is usually not NO responsive. We suggest that cGMP plays a role in, or reflects, the early stages of communication between a target and specific innervating neurons.

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme : the active site H103W mutation restores ADP-ribosyl cyclase activity†

PubMed Central

Kuhn, Isabelle; Kellenberger, Esther; Rognan, Didier; Lund, Frances E.; Muller-Steffner, Hélène; Schuber, Francis

2008-01-01

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes which are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products, i.e., a value larger than observed for other members of this family such as CD38. Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provide new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge however in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR. PMID:17002287

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. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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

Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

PubMed

Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

2017-08-31

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

PubMed Central

Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

2012-01-01

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

Cardiovascular and adenylate cyclase stimulating effects of colforsin daropate, a water-soluble forskolin derivative, compared with those of isoproterenol, dopamine and dobutamine.

PubMed

Yoneyama, Masahiko; Sugiyama, Atsushi; Satoh, Yoshioki; Takahara, Akira; Nakamura, Yuji; Hashimoto, Keitaro

2002-12-01

Colforsin daropate is a recently developed water-soluble derivative of forskolin that directly stimulates adenylate cyclase, unlike the catecholamines. The chronotropic, inotropic and coronary vasodilator actions of colforsin daropate were compared with those of isoproterenol, dopamine and dobutamine, using canine isolated, blood-perfused heart preparations. The stimulating effect of each drug on adenylate cyclase activity was also assessed. Colforsin daropate, as well as each of the catecholamines, exerted positive chronotropic, inotropic and coronary vasodilator actions. The order of selectivity for the cardiovascular variables of colforsin daropate was coronary vasodilation > positive inotropy > positive chronotropy; whereas that of isoproterenol, dopamine and dobutamine was positive inotropy > coronary vasodilation > positive chronotropy. Thus, a marked characteristic of colforsin daropate is its potent coronary vasodilator action. On the other hand, each drug significantly increased the adenylate cyclase activity in a dose-related manner: colforsin daropate > isoproterenol > dopamine = dobutamine. These results suggest that colforsin daropate may be preferable in the treatment of severe heart failure where the coronary blood flow is reduced and beta-adrenoceptor-dependent signal transduction pathway is down-regulated.

Adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in Brazilian children.

PubMed

Teixeira, Helena M P; Alcantara-Neves, Neuza M; Barreto, Maurício; Figueiredo, Camila A; Costa, Ryan S

2017-02-01

Asthma is a chronic inflammatory disease of the respiratory tract. This heterogeneous disease is caused by the interaction of interindividual genetic variability and environmental factors. The gene adenylyl cyclase type 9 (ADCY9) encodes a protein called adenylyl cyclase (AC), responsible for producing the second messenger cyclic AMP (cAMP). cAMP is produced by T regulatory cells and is involved in the down-regulation of T effector cells. Failures in cAMP production may be related to an imbalance in the regulatory immune response, leading to immune-mediated diseases, such as allergic disorders. The aim of this study was to investigate how polymorphisms in the ADCY9 are associated with asthma and allergic markers. The study comprised 1309 subjects from the SCAALA (Social Changes Asthma and Allergy in Latin America) program. Genotyping was accomplished using the Illumina 2.5 Human Omni bead chip. Logistic regression was used to assess the association between allergy markers and ADCY9 variation in PLINK 1.07 software with adjustments for sex, age, helminth infection and ancestry markers. The ADCY9 candidate gene was associated with different phenotypes, such as asthma, specific IgE, skin prick test, and cytokine production. Among 133 markers analyzed, 29 SNPs where associated with asthma and allergic markers in silico analysis revealed the functional impact of the 6 SNPs on ADCY9 expression. It can be concluded that polymorphisms in the ADCY9 gene are significantly associated with asthma and/or allergy markers. We believe that such polymorphisms may lead to increased expression of adenylyl cyclase with a consequent increase in immunoregulatory activity. Therefore, these SNPs may offer an impact on the occurrence of these conditions in admixture population from countries such as Brazil. Copyright © 2017 Elsevier Ltd. All rights reserved.

Soluble Adenylyl Cyclase of Sea Urchin Spermatozoa

PubMed Central

Vacquier, Victor D.; Loza-Huerta, Arlet; García-Rincón, Juan; Darszon, Alberto; Beltrán, Carmen

2014-01-01

Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are amongst the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO3- it is able to translate CO2/HCO3-/pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130 years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. PMID:25064590

A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

PubMed Central

Arrach, Nabil; Fernández-Martín, Rafael; Cerdá-Olmedo, Enrique; Avalos, Javier

2001-01-01

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces. PMID:11172012

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

PubMed

Kaufmann, R; Schöneberg, T; Henklein, P; Meyer, R; Martin, H; Ott, T

1995-07-01

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

Solubilization and other studies on adenylate cyclase of baker's yeast.

PubMed Central

Varimo, K; Londesborough, J

1976-01-01

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000. PMID:793584

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

PubMed Central

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

2014-01-01

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.—Hess, K. C., Liu, J., Manfredi, G., Mühlschlegel, F. A., Buck, J., Levin, L. R., Barrientos, A. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. PMID:25002117

The low-dose combination preparation Vertigoheel activates cyclic nucleotide pathways and stimulates vasorelaxation.

PubMed

Heinle, H; Tober, C; Zhang, D; Jäggi, R; Kuebler, W M

2010-01-01

Vertigo of various and often unknown aetiologies has been associated with and attributed to impaired microvascular perfusion in the inner ear or the vertebrobasilar system. Vertigoheel is a low-dose combination preparation of proven value in the symptomatic treatment of vertigo. In the present study we tested the hypothesis that Vertigoheel's anti-vertiginous properties may in part be due to a vasodilatory effect exerted via stimulation of the adenylate and/or guanylate cyclase pathways. Thus, the influence of Vertigoheel or its single constituents on synthesis and degradation of cyclic nucleotides was measured. Furthermore, vessel myography was used to observe the effect of Vertigoheel on the vasoreactivity of rat carotid arteries. Vertigoheel and one of its constituents, Anamirta cocculus, stimulated adenylate cyclase activity, while another constituent, Conium maculatum, inhibited phosphodiesterase 5, suggesting that the individual constituents of Vertigoheel contribute differentially to a synergistic stimulation of cyclic nucleotide signalling pathways. In rat carotid artery rings, Vertigoheel counteracted phenylephrine-induced tonic vasoconstriction. The present data demonstrate a vasorelaxant effect of Vertigoheel that goes along with a synergistic stimulation of cyclic nucleotide pathways and may provide a mechanistic basis for the documented anti-vertiginous effects of this combination preparation.

Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP.

PubMed Central

Barsony, J; Marx, S J

1990-01-01

We developed a method for cAMP and cGMP immunocytology based upon fixation by microwave irradiation. Fixation by microwave irradiation prevented three problems found with other fixation methods: nucleotide loss from cells, nucleotide diffusion within cells, and chemical modification of immunologic epitopes. Six agonists (four that stimulate adenylate cyclase and two that stimulate guanylate cyclase) produced cAMP or cGMP accumulation patterns that were agonist-specific, dose-dependent, detectable at physiologic concentrations of hormone, and time-dependent within 15 sec to 30 min. cAMP accumulation after 1 mM forskolin was greatest in the nucleus. Isoproterenol, prostaglandin E2, or calcitonin caused initial accumulation of cAMP along the plasma membrane, but later accumulation was greater in the cytoplasm. With calcitonin the later accumulation of cAMP was selectively perinuclear and along the nuclear membrane. Sodium nitroprusside stimulated cGMP accumulation diffusely throughout the cytoplasm. Atrial natriuretic peptide initiated cGMP accumulation near the plasma membrane, and cGMP accumulation moved from there into the cytoplasm. In conclusion, microwave irradiation preserved cell structure and allowed visualization of expected as well as unsuspected changes in intracellular accumulation patterns of cAMP and cGMP. Images PMID:2153973

THE SHARK RECTAL GLAND MODEL: A CHAMPION OF RECEPTOR MEDIATED CHLORIDE SECRETION THROUGH CFTR

PubMed Central

FORREST, JOHN N.

2016-01-01

The dogfish shark salt gland was predicted by Smith and discovered by Burger at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine. It is an epithelial organ in the intestine composed of tubules that serve a single function: the secretion of hypertonic NaCl. Many G protein receptors are present on the basolateral surface of these tubules, including stimulatory receptors for vasoactive intestinal peptide, adenosine A2, growth hormone releasing hormone, and inhibitory receptors for somatostatin and adenosine A1. An entirely different class of stimulatory receptors is present as C-type natriuretic peptide receptors. Each stimulatory receptor evokes powerful NaCl secretion. G protein receptors bind to Gαs to activate the catalytic unit of adenylate cyclase to form cyclic adenosine monophosphate (cAMP) and protein kinase A that phosphorylates the regulatory domain of cystic fibrosis transmembrane conductance regulator, opening the channel. The C-type natriuretic peptide receptor stimulates by activating guanylate cyclase and endogenous cyclic guanosine monophosphate which inhibits type 3 phosphodiesterase, the enzyme that breaks down cAMP, thereby elevating cAMP and activating the protein kinase A pathway. PMID:28066051

Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, S.Q.; Ren, Y.F.; Alam, B.S.

1987-05-01

The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mgmore » protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.« less

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

PubMed

Dix, C J; Habberfield, A D; Cooke, B A

1984-06-15

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig

Current developments in pharmacological therapeutics for chronic constipation

PubMed Central

Jiang, Chunhuan; Xu, Qinglong; Wen, Xiaoan; Sun, Hongbin

2015-01-01

Chronic constipation is a common gastrointestinal disease severely affecting the patient׳s quality of life. The traditional treatment of constipation is the use of laxatives. Recently, several new drugs including lubiprostone, linaclotide and prucalopride have been approved for treatment of chronic constipation. However, a significant unmet medical need still remains, particularly among those patients achieving poor results by current therapies. The 5-HT4 receptor modulators velusetrag and naronapride, the guanylate cyclase C agonist plecanatide and the ileal bile acid transporter inhibitor elobixibat are recognized as the most promising drugs under investigation. Herein, we give a comprehensive review on the pharmacological therapeutics for the treatment of chronic constipation, with the purpose of reflecting the drug development trends in this field. PMID:26579459

Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.

PubMed

Pérez-Sala, D; Cernuda-Morollón, E; Díaz-Cazorla, M; Rodríguez-Pascual, F; Lamas, S

2001-03-01

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1beta plus tumor necrosis factor (TNF)-alpha reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1beta/TNF-alpha-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.

Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

PubMed

Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

1996-05-31

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

Nitrosopersulfide (SSNO(-)) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide.

PubMed

Cortese-Krott, Miriam M; Fernandez, Bernadette O; Santos, José L T; Mergia, Evanthia; Grman, Marian; Nagy, Péter; Kelm, Malte; Butler, Anthony; Feelisch, Martin

2014-01-01

Sulfide salts are known to promote the release of nitric oxide (NO) from S-nitrosothiols and potentiate their vasorelaxant activity, but much of the cross-talk between hydrogen sulfide and NO is believed to occur via functional interactions of cell regulatory elements such as phosphodiesterases. Using RFL-6 cells as an NO reporter system we sought to investigate whether sulfide can also modulate nitrosothiol-mediated soluble guanylyl cyclase (sGC) activation following direct chemical interaction. We find a U-shaped dose response relationship where low sulfide concentrations attenuate sGC stimulation by S-nitrosopenicillamine (SNAP) and cyclic GMP levels are restored at equimolar ratios. Similar results are observed when intracellular sulfide levels are raised by pre-incubation with the sulfide donor, GYY4137. The outcome of direct sulfide/nitrosothiol interactions also critically depends on molar reactant ratios and is accompanied by oxygen consumption. With sulfide in excess, a 'yellow compound' accumulates that is indistinguishable from the product of solid-phase transnitrosation of either hydrosulfide or hydrodisulfide and assigned to be nitrosopersulfide (perthionitrite, SSNO(-); λ max 412 nm in aqueous buffers, pH 7.4; 448 nm in DMF). Time-resolved chemiluminescence and UV-visible spectroscopy analyses suggest that its generation is preceded by formation of the short-lived NO-donor, thionitrite (SNO(-)). In contrast to the latter, SSNO(-) is rather stable at physiological pH and generates both NO and polysulfides on decomposition, resulting in sustained potentiation of SNAP-induced sGC stimulation. Thus, sulfide reacts with nitrosothiols to form multiple bioactive products; SSNO(-) rather than SNO(-) may account for some of the longer-lived effects of nitrosothiols and contribute to sulfide and NO signaling.

Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

PubMed Central

Huang, Ngan F.; Fleissner, Felix; Sun, John

2010-01-01

Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (l-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, l-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of l-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of l-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by l-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function. PMID:20064011

Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

PubMed Central

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

PubMed

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J K; Schubert, Dirk W; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh

Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved throughmore » its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex

Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

PubMed

Roa, Jinae N; Tresguerres, Martin

2016-08-01

Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology. Copyright © 2016 the American Physiological Society.

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells.

PubMed

Ding, K H; Latimer, A J; Abdel-Latif, A A

1999-01-01

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.

(/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, S.Q.; Ren, Y.F.; Alam, B.S.

1988-03-01

The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oilmore » than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.« less

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.

PubMed

Wu, Vincent W; Dana, Craig M; Iavarone, Anthony T; Clark, Douglas S; Glass, N Louise

2017-01-17

The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. Pyroglutamate modification is the post-translational conversion of N-terminal glutamine or glutamate into a cyclized amino acid derivative. This modification is well studied in animal systems but poorly explored in fungal systems. In Neurospora crassa, we show that this modification takes place in the ER and is catalyzed by two well-conserved enzymes, ubiquitously conserved throughout the fungal kingdom. We

Functional EF-Hands in Neuronal Calcium Sensor GCAP2 Determine Its Phosphorylation State and Subcellular Distribution In Vivo, and Are Essential for Photoreceptor Cell Integrity

PubMed Central

Rosa, Jose Luis; Chen, Jeannie; Méndez, Ana

2014-01-01

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF−GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF−GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in �

The Growth of SGC-7901 Tumor Xenografts Was Suppressed by Chinese Bayberry Anthocyanin Extract through Upregulating KLF6 Gene Expression.

PubMed

Wang, Yue; Zhang, Xia-Nan; Xie, Wen-Hua; Zheng, Yi-Xiong; Cao, Jin-Ping; Cao, Pei-Rang; Chen, Qing-Jun; Li, Xian; Sun, Chong-de

2016-09-27

To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit ( Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G (cyanidin-3-glucoside, an anthocyanin) significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo.

The Growth of SGC-7901 Tumor Xenografts Was Suppressed by Chinese Bayberry Anthocyanin Extract through Upregulating KLF6 Gene Expression

PubMed Central

Wang, Yue; Zhang, Xia-nan; Xie, Wen-hua; Zheng, Yi-xiong; Cao, Jin-ping; Cao, Pei-rang; Chen, Qing-jun; Li, Xian; Sun, Chong-de

2016-01-01

To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit (Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G (cyanidin-3-glucoside, an anthocyanin) significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo. PMID:27690088

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation

PubMed Central

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-01-01

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant. PMID:25672880

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation.

PubMed

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-02-12

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant.

CO2/HCO3−- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

PubMed Central

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

2013-01-01

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. PMID:24100033

Association of Adenylate Cyclase 10 (ADCY10) Polymorphisms and Bone Mineral Density in Healthy Adults

PubMed Central

Ichikawa, Shoji; Koller, Daniel L.; Curry, Leah R.; Lai, Dongbing; Xuei, Xiaoling; Edenberg, Howard J.; Hui, Siu L.; Peacock, Munro; Foroud, Tatiana; Econs, Michael J.

2010-01-01

Phenotypic variation in bone mineral density (BMD) among healthy adults is influenced by both genetic and environmental factors. Genetic sequence variations in the adenylate cyclase 10 (ADCY10) gene, which is also called soluble adenylate cyclase, have previously been reported to be associated with low spinal BMD in hypercalciuric patients. Since ADCY10 is located in the region linked to spinal BMD in our previous linkage analysis, we tested whether polymorphisms in this gene are also associated with normal BMD variation in healthy adults. Sixteen single nucleotide polymorphisms (SNPs) distributed throughout ADCY10 were genotyped in two healthy groups of American whites: 1,692 premenopausal women and 715 men. Statistical analyses were performed in the two groups to test for association between these SNPs and femoral neck and lumbar spine areal BMD. We observed significant evidence of association (p<0.01) with one SNP each in men and women. Genotypes at these SNPs accounted for less than 1% of hip BMD variation in men, but 1.5% of spinal BMD in women. However, adjacent SNPs did not corroborate the association in either males or females. In conclusion, we found a modest association between an ADCY10 polymorphism and spinal areal BMD in premenopausal white women. PMID:19093065

M‐Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics

PubMed Central

McKie, Paul M.; Cataliotti, Alessandro; Ichiki, Tomoko; Sangaralingham, S. Jeson; Chen, Horng H.; Burnett, John C.

2014-01-01

Background Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M‐atrial natriuretic peptide (M‐ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator. Methods and Results HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M‐ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M‐ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M‐ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M‐ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation. Conclusions Our results advance the differential cardiorenal actions of pGC (M‐ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M‐ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal. PMID:24385449

M-atrial natriuretic peptide and nitroglycerin in a canine model of experimental acute hypertensive heart failure: differential actions of 2 cGMP activating therapeutics.

PubMed

McKie, Paul M; Cataliotti, Alessandro; Ichiki, Tomoko; Sangaralingham, S Jeson; Chen, Horng H; Burnett, John C

2014-01-02

Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator. HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M-ANP (139 ± 4 to 118 ± 3 mm Hg, P<0.05) and nitroglycerin (137 ± 3 to 116 ± 4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M-ANP (12 ± 2 to 6 ± 2 mm Hg, P<0.05) and nitroglycerin (12 ± 1 to 6 ± 1 mm Hg, P<0.05). M-ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38 ± 4 to 53 ± 5 mL/min), renal blood flow (132 ± 18 to 236 ± 23 mL/min), and natriuresis (11 ± 4 to 689 ± 37 mEq/min) and also inhibited aldosterone activation (32 ± 3 to 23 ± 2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation. Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Detection and description of various stores of nitric oxide store in vascular wall.

PubMed

Vlasova, M A; Vanin, A F; Muller, B; Smirin, B V; Malyshev, I Yu; Manukhina, E B

2003-09-01

We analyzed the possibility of the existence of various NO pools in the vascular wall. Incubation of isolated rat aorta with dinitrosyl iron complex (NO donor) led to the formation of NO stores in the vascular wall detected by vascular relaxation response induced by diethyldithiocarbamate and N-acetylcysteine. Comparison of the effects of successive application of diethyldithiocarbamate and N-acetylcysteine revealed two NO pools (one pool responded to both agents, while other responded only to N-acetylcysteine). Inhibition of guanylate cyclase with methylene blue abolished the response to diethyldithiocarbamate, while the reaction to N-acetylcysteine decreased by the value, corresponding to diethyldithiocarbamate-dependent relaxation. It is hypothesized that in the vascular wall NO is stored in the form protein-bound dinitrosyl iron complexes and S-nitrosothiols in hydrophilic and hydrophobic cell compartments.

A Chemoreceptor That Detects Molecular Carbon Dioxide*

PubMed Central

Smith, Ewan St. John; Martinez-Velazquez, Luis; Ringstad, Niels

2013-01-01

Animals from diverse phyla possess neurons that are activated by the product of aerobic respiration, CO2. It has long been thought that such neurons primarily detect the CO2 metabolites protons and bicarbonate. We have determined the chemical tuning of isolated CO2 chemosensory BAG neurons of the nematode Caenorhabditis elegans. We show that BAG neurons are principally tuned to detect molecular CO2, although they can be activated by acid stimuli. One component of the BAG transduction pathway, the receptor-type guanylate cyclase GCY-9, suffices to confer cellular sensitivity to both molecular CO2 and acid, indicating that it is a bifunctional chemoreceptor. We speculate that in other animals, receptors similarly capable of detecting molecular CO2 might mediate effects of CO2 on neural circuits and behavior. PMID:24240097

Nitric oxide (NO.) stabilizes whereas nitrosonium (NO+) enhances filopodial outgrowth by rat retinal ganglion cells in vitro.

PubMed

Cheung, W S; Bhan, I; Lipton, S A

2000-06-16

Recent observations suggest that nitric oxide (NO(.)) can increase or decrease growth cone motility. Here, these apparently paradoxical results are explained by distinct actions of different NO-related species. Filopodial morphology of 223 rat retinal ganglion cells was monitored under computer-enhanced video microscopy in the presence of NO synthase (NOS) substrates or inhibitors, donors of specific NO-related species, and membrane-permeant cyclic nucleotide analogs. Physiological NOS activity induced filopodial outgrowth, whereas inhibition of NOS stabilized filopodia. Similar to NOS, nitrosonium (NO(+) transfer) and peroxynitrite (ONOO(-)), which can regulate the activity of growth-associated proteins by S-nitrosylation and oxidation, respectively, induced filopodial outgrowth. In contrast, NO(.), which stimulates guanylate cyclase to increase cGMP, stabilized filopodial activity. Thus disparate NO-related species may offer a dynamic process of filopodial growth regulation.

The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons.

PubMed

Uchida, Okiko; Nakano, Hiroyuki; Koga, Makoto; Ohshima, Yasumi

2003-04-01

Chemotaxis to water-soluble chemicals such as NaCl is an important behavior of C. elegans when seeking food. ASE chemosensory neurons have a major role in this behavior. We show that che-1, defined by chemotaxis defects, encodes a zinc-finger protein similar to the GLASS transcription factor required for photoreceptor cell differentiation in Drosophila, and that che-1 is essential for specification and function of ASE neurons. Expression of a che-1::gfp fusion construct was predominant in ASE. In che-1 mutants, expression of genes characterizing ASE such as seven-transmembrane receptors, guanylate cyclases and a cyclic-nucleotide gated channel is lost. Ectopic expression of che-1 cDNA induced expression of ASE-specific marker genes, a dye-filling defect in neurons other than ASE and dauer formation.

The temperature-dependence of adenylate cyclase from baker's yeast.

PubMed Central

Londesborough, J; Varimo, K

1979-01-01

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism. PMID:391221

Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

2018-03-01

Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

NASA Technical Reports Server (NTRS)

Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

1995-01-01

1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols.

PubMed

Bautista-Niño, Paula K; van der Stel, Marien; Batenburg, Wendy W; de Vries, René; Roks, Anton J M; Danser, A H Jan

2018-05-15

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H 2 O 2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H 2 O 2 -, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H 2 O 2 , but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H 2 O 2 , were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na + -K + ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na + -K + ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H 2 O 2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

PubMed

Ronchetti, Sonia A; Machiavelli, Leticia I; Quinteros, Fernanda A; Duvilanski, Beatriz H; Cabilla, Jimena P

2016-01-01

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary.

Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen

PubMed Central

Quinteros, Fernanda A.; Duvilanski, Beatriz H.; Cabilla, Jimena P.

2016-01-01

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary. PMID:27611913

Selectivity of Vibrio cholerae H-NOX for Gaseous Ligands Follows “Sliding Scale Rule” Hypothesis

PubMed Central

Wu, Gang; Liu, Wen; Berka, Vladimir; Tsai, Ah-lim

2014-01-01

Vc H-NOX (or VCA0720) is an H-NOX (heme-nitric oxide and oxygen binding) protein from facultative aerobic bacterium Vibrio cholerae. It shares significant sequence homology with soluble guanylyl cyclase (sGC), a NO sensor protein commonly found in animals. Similar to sGC, Vc H-NOX binds strongly to NO and CO with affinities of 0.27 nM and 0.77 μM, respectively, but weakly to O2. When positioned in “sliding scale” plot {Tsai, A.-L. et. al. (2012) Biochemistry, 51, pp172-86}, the line connecting logKD(NO) and logKD(CO) of Vc H-NOX is almost superimposable with that of Ns H-NOX. Therefore, the measured affinities and kinetic parameters of gaseous ligands to Vc H-NOX provide more evidence to validate the “sliding scale rule” hypothesis. Like sGC, Vc H-NOX binds NO in multiple steps, forming first a 6-coordinate heme-NO complex with a rate of 1.1 × 109 M−1s−1, and then converts to a 5c heme-NO complex at a rate also dependent on [NO]. Although the formation of oxyferrous Vc H-NOX is not detectable under normal atmospheric oxygen level, ferrous Vc H-NOX is oxidized to ferric form at a rate of 0.06 s−1 when mixed with O2. Ferric Vc H-NOX exists as a mixture of high- and low-spin states and is influenced by binding to different ligands. Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lay the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX. PMID:24351060

Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

PubMed

Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

2015-01-01

Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

The Type 3 Adenylyl Cyclase is Required for Novel Object Learning and Extinction of Contextual Memory: Role of cAMP Signaling in Primary Cilia

PubMed Central

Wang, Zhenshan; Phan, Trongha; Storm, Daniel R.

2011-01-01

Although primary cilia are found on neurons throughout the brain, their physiological function remains elusive. Human ciliopathies are associated with cognition defects and transgenic mice lacking proteins expressed in primary cilia exhibit defects in learning and memory. Recently, it was reported that mice lacking the G-protein coupling receptor somatostatin receptor-3 (SSTR3), a protein expressed predominately in the primary cilia of neurons, have defective memory for novel object recognition and lower cAMP levels in the brain. Since SSTR3 is coupled to regulation of adenylyl cyclase this suggests that adenylyl cyclase activity in primary cilia of CNS neurons may be critical for some forms of learning and memory. Because the type 3 adenylyl cyclase (AC3) is expressed in primary cilia of hippocampal neurons, we examined AC3−/− mice for several forms of learning and memory. Here, we report that AC3−/− mice show no short-term memory for novel objects and fail to exhibit extinction of contextual fear conditioning. They also show impaired learning and memory for temporally dissociated passive avoidance (TDPA). Since AC3 is exclusively expressed in primary cilia we conclude that cAMP signals generated within primary cilia contribute to some forms of learning and memory including extinction of contextual fear conditioning. PMID:21490195

The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia.

PubMed

Wang, Zhenshan; Phan, Trongha; Storm, Daniel R

2011-04-13

Although primary cilia are found on neurons throughout the brain, their physiological function remains elusive. Human ciliopathies are associated with cognition defects, and transgenic mice lacking proteins expressed in primary cilia exhibit defects in learning and memory. Recently, it was reported that mice lacking the G-protein-coupling receptor somatostatin receptor-3 (SSTR3), a protein expressed predominately in the primary cilia of neurons, have defective memory for novel object recognition and lower cAMP levels in the brain. Since SSTR3 is coupled to regulation of adenylyl cyclase, this suggests that adenylyl cyclase activity in primary cilia of CNS neurons may be critical for some forms of learning and memory. Because the type 3 adenylyl cyclase (AC3) is expressed in primary cilia of hippocampal neurons, we examined AC3(-/-) mice for several forms of learning and memory. Here, we report that AC3(-/-) mice show no short-term memory for novel objects and fail to exhibit extinction of contextual fear conditioning. They also show impaired learning and memory for temporally dissociative passive avoidance. Since AC3 is exclusively expressed in primary cilia, we conclude that cAMP signals generated within primary cilia contribute to some forms of learning and memory, including extinction of contextual fear conditioning.

Guanylate-binding protein-1 is a potential new therapeutic target for triple-negative breast cancer.

PubMed

Quintero, Melissa; Adamoski, Douglas; Reis, Larissa Menezes Dos; Ascenção, Carolline Fernanda Rodrigues; Oliveira, Krishina Ratna Sousa de; Gonçalves, Kaliandra de Almeida; Dias, Marília Meira; Carazzolle, Marcelo Falsarella; Dias, Sandra Martha Gomes

2017-11-07

Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression (ESR and PGR, respectively) and an absence of human epithelial growth factor receptor (ERBB2) amplification. Approximately 15-20% of breast malignancies are TNBC. Patients with TNBC often have an unfavorable prognosis. In addition, TNBC represents an important clinical challenge since it does not respond to hormone therapy. In this work, we integrated high-throughput mRNA sequencing (RNA-Seq) data from normal and tumor tissues (obtained from The Cancer Genome Atlas, TCGA) and cell lines obtained through in-house sequencing or available from the Gene Expression Omnibus (GEO) to generate a unified list of differentially expressed (DE) genes. Methylome and proteomic data were integrated to our analysis to give further support to our findings. Genes that were overexpressed in TNBC were then curated to retain new potentially druggable targets based on in silico analysis. Knocking-down was used to assess gene importance for TNBC cell proliferation. Our pipeline analysis generated a list of 243 potential new targets for treating TNBC. We finally demonstrated that knock-down of Guanylate-Binding Protein 1 (GBP1 ), one of the candidate genes, selectively affected the growth of TNBC cell lines. Moreover, we showed that GBP1 expression was controlled by epidermal growth factor receptor (EGFR) in breast cancer cell lines. We propose that GBP1 is a new potential druggable therapeutic target for treating TNBC with enhanced EGFR expression.

Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Ping; Zhu, Xueping; Jin, Wei

Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygenmore » species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its

Synergistic effects of BAY 60-4552 and vardenafil on relaxation of corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure.

PubMed

Albersen, Maarten; Linsen, Loes; Tinel, Hanna; Sandner, Peter; Van Renterghem, Koenraad

2013-05-01

Overall efficacy rates of phosphodiesterase type 5 inhibitors (PDE5-i) for erectile dysfunction (ED) are 60-70%. PDE5-i treatment failures currently have to resort to invasive treatment options for restoration of erectile function. AIMS.: To assess changes in the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase (PKG) pathway in human corpus cavernosum (HCC) of PDE5-i nonresponders compared with healthy controls. To evaluate the effects of BAY 60-4552, a stimulator of soluble guanylate cyclase (sGC), and vardenafil on relaxation of HCC strips from PDE5-i nonresponders. mRNA expression, morphological localization of the NO/cGMP/PKG pathway, and relaxant capacity of both compounds alone or combined. Analysis of variance, t-test or Mann-Whitney test based upon number of groups and normality of data. HCC tissues were harvested after consent from individuals undergoing penile prosthesis implantation (patients) and potent patients undergoing transurethral surgery (healthy controls, needle biopsy). HCC tissues of patients were compared with those of healthy controls for the expression of mRNA coding for PDE5A, eNOS, PKGα1, PKG2, sGCα1, sGCα2, sGCβ1, sGCβ2, α-smooth muscle actin (aSMA) and β-actin by quantitative polymerase chain reaction (qPCR). The respective proteins were localized using immunofluorescence. Tissue strips of patients were precontracted with phenylepinephrine followed by incubation with 1 μM of either vardenafil or BAY 60-4552, or both simultaneously. The main targets in the NO/cGMP/sGC pathway were downregulated in PDE5-i nonresponders. The pathway was morphologically located to HCC smooth muscle, of which the overall content was preserved in ED patients based on aSMA expression. BAY 60-4552 and vardenafil have synergistic effects on relaxation of HCC of PDE5-i nonresponders. The main limitation is the small amount of control tissue precluding functional testing on these samples. Despite downregulation of the NO

Isolation and Characterization of a Lycopene ε-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light

PubMed Central

Cordero, Baldo F.; Couso, Inmaculada; Leon, Rosa; Rodriguez, Herminia; Vargas, Maria Angeles

2012-01-01

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress. PMID:23118722

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…

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

PubMed Central

Rahman, Safikur; Deep, Shashank; Sau, Apurba Kumar

2012-01-01

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1. PMID:22859948

High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

PubMed

Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

2014-01-17

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

PubMed

Bijvelds, Marcel J C; Loos, Michaela; Bronsveld, Inez; Hellemans, Ann; Bongartz, Jean-Pierre; Ver Donck, Luc; Cox, Eric; de Jonge, Hugo R; Schuurkes, Jan A J; De Maeyer, Joris H

2015-12-01

Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer

PubMed Central

Blomain, Erik S.; Merlino, Dante J.; Pattison, Amanda M.; Snook, Adam E.

2016-01-01

Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity

Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-Ring cyclization and deprotonation reactions.

PubMed

Wu, Tung-Kung; Liu, Yuan-Ting; Chiu, Feng-Hsuan; Chang, Cheng-Hsiang

2006-10-12

[reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

Cysteine-Zn2+ complexes: unique molecular switches for inducible nitric oxide synthase-derived NO.

PubMed

Kröncke, K D

2001-11-01

Nitric oxide (NO) in the low nanomolar range acts as a transcellular messenger molecule to initiate regulatory and physiological responses in nearby target cells via binding to the soluble guanylate cyclase heme moiety. Higher NO concentrations, as synthesized by the inducible NO synthase (iNOS) during inflammatory processes, show additional effects: NO may react with O2, yielding nitrogen oxides like N2O3 that are able to nitrosate thiols. A variety of proteins involved in very different functions of the cell contain cysteine-Zn2+ complexes. Effects of NO on different proteins containing cysteine-Zn2+ domains and playing essential roles during transcription, protein folding, and proteolysis are discussed. It is suggested that iNOS-derived NO acts as a signal molecule targeting cysteine-Zn2+ linkages, thus enabling cells to react toward nitrosative stress.

NITRIC OXIDE, MITOCHONDRIAL HYPERPOLARIZATION AND T-CELL ACTIVATION

PubMed Central

Nagy, Gyorgy; Koncz, Agnes; Fernandez, David; Perl, Andras

2007-01-01

T lymphocyte activation is associated with nitric oxide (NO) production that plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both T lymphocyte activation and apoptosis, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus (SLE) induces mitochondrial biogenesis and alters Ca2+ signaling. Thus, while NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity. PMID:17462531

Type 1 Adenylyl Cyclase is Essential for Maintenance of Remote Contextual Fear Memory

PubMed Central

Shan, Qiang; Chan, Guy C.-K.; Storm, Daniel R.

2008-01-01

Although molecular mechanisms for hippocampus-dependent memory have been extensively studied, much less is known about signaling events important for remote memory. Here we report that mice lacking type 1 adenylyl cyclase (AC1) are able to establish and retrieve remote contextual memory but unable to sustain it as long as wild type mice. Interestingly, mice over-expressing AC1 show superior remote contextual memory even though they exhibit normal hippocampus-dependent contextual memory. These data illustrate that calcium coupling to cAMP contributes to the stability of remote memory and identifies AC1 as a potential drug target site to improve long-term remote memory. PMID:19036980

Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronin, M.J.; Evans, W.S.; Rogol, A.D.

1986-08-01

Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplifiedmore » the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.« less

Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms.

PubMed

Bradbury, Louis M T; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J; Wurtzel, Eleanore T

2012-07-03

In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784-11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that CruP aids in preventing accumulation of reactive oxygen species (ROS), thereby reducing accumulation of β-carotene-5,6-epoxide, a ROS-catalyzed autoxidation product, and inhibiting accumulation of anthocyanins, which are known chemical indicators of ROS. Plants with a nonfunctional cruP accumulate substantially higher levels of ROS and β-carotene-5,6-epoxide in green tissues. Plants overexpressing cruP show reduced levels of ROS, β-carotene-5,6-epoxide, and anthocyanins. The observed up-regulation of cruP transcripts under photoinhibitory and lipid peroxidation-inducing conditions, such as high light stress, cold stress, anoxia, and low levels of CO(2), fits with a role for CruP in mitigating the effects of ROS. Phylogenetic distribution of CruP in prokaryotes showed that the gene is only present in cyanobacteria that live in habitats characterized by large variation in temperature and inorganic carbon availability. Therefore, CruP represents a unique target for developing resilient plants and algae needed to supply food and biofuels in the face of global climate change.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

PubMed Central

Wang, Xianzhe; Maynard, Jennifer A.

2015-01-01

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

PubMed Central

Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

1998-01-01

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

PubMed Central

Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

2012-01-01

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer.

PubMed

Blomain, Erik S; Merlino, Dante J; Pattison, Amanda M; Snook, Adam E; Waldman, Scott A

2016-09-01

Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity

Long-range allosteric signaling in red light–regulated diguanylyl cyclases

PubMed Central

Gourinchas, Geoffrey; Etzl, Stefan; Göbl, Christoph; Vide, Uršula; Madl, Tobias; Winkler, Andreas

2017-01-01

Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light–sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate–producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light–regulated optogenetic tools. PMID:28275738

The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT.

PubMed

Sun, Yi-Cheng; Guo, Xiao-Peng; Hinnebusch, B Joseph; Darby, Creg

2012-04-01

Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

Isolation and functional characterization of a lycopene β-cyclase gene that controls fruit colour of papaya (Carica papaya L.)

PubMed Central

Devitt, Luke C.; Fanning, Kent; Dietzgen, Ralf G.; Holton, Timothy A.

2010-01-01

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to β-carotene (yellow) is catalysed by lycopene β-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene β-cyclases (lcy-β1 and lcy-β2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-β2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-β1 and lcy-β2 genes is similar and low in leaves, but lcy-β2 expression increases markedly in ripe fruit. Isolation of the lcy-β2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties. PMID:19887502

Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

PubMed

Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

2010-01-01

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

S-nitrosothiol transport via PEPT2 mediates biological effects of nitric oxide gas exposure in macrophages.

PubMed

Brahmajothi, Mulugu V; Sun, Natalie Z; Auten, Richard L

2013-02-01

The pharmacological effects of nitric oxide (NO) administered as a gas are dependent on the conversion to S-nitrosocysteine, and as such are largely mediated by the L-type amino-acid transporters (LATs) in several cell types. The dipeptide transporter PEPT2 has been proposed as a second route for S-nitrosothiol (SNO) transport, but this has never been demonstrated. Because NO governs important immune functions in alveolar macrophages, we exposed rat alveolar macrophages (primary and NR8383 cells) to NO gas at the air-liquid interface ± LPS stimulation in the presence of PEPT2 substrate Cys-Gly (or the LAT substrate L-Cys) ± transporter competitors. We found that SNO uptake and NO-dependent actions, such as the activation of soluble guanylyl cyclase (sGC), the augmentation of sGC-dependent filamentous actin (F-actin) polymerization, phagocytosis, and the inhibition of NF-κB activation, were significantly augmented by the addition of Cys-Gly in a manner dependent on PEPT2 transport. We found parallel (and greater) effects that were dependent on LAT transport. The contribution of cystine/cysteine shuttling via system x cystine transporter (xCT) to SNO uptake was relatively minor. The observed effects were unaffected by NO synthase inhibition. The NO gas treatment of alveolar macrophages increased SNO uptake, the activation of sGC, F-actin polymerization, and phagocytosis, and inhibited NF-κB activation, in a manner dependent on SNO transport via PEPT2, as well as via LAT.

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.

Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

NASA Technical Reports Server (NTRS)

Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

1999-01-01

The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

Effects of the nitric oxide donor JS-K on the blood-tumor barrier and on orthotopic U87 rat gliomas assessed by MRI

PubMed Central

Weidensteiner, Claudia; Reichardt, Wilfried; Shami, Paul J.; Saavedra, Joseph E.; Keefer, Larry K.; Baumer, Brunhilde; Werres, Anna; Jasinski, Robert; Osterberg, Nadja; Weyerbrock, Astrid

2013-01-01

Nitric oxide (NO) released from NO donors can be cytotoxic in tumor cells and can enhance the transport of drugs into brain tumors by altering blood-tumor barrier permeability. The NO donor JS-K [O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] releases NO upon enzymatic activation selectively in cells overexpressing glutathione-S-transferases (GSTs) such as gliomas. Thus, JS-K-dependent NO effects - especially on cell viability and vascular permeability - were investigated in U87 glioma cells in vitro and in an orthotopic U87 xenograft model in vivo by magnetic resonance imaging (MRI). In vitro experiments showed dose-dependent antiproliferative and cytotoxic effects in U87 cells. In addition, treatment of U87 cells with JS-K resulted in a dose-dependent activation of soluble guanylate cyclase and intracellular accumulation of cyclic guanosine monophosphate (cGMP) which was irreversibly inhibited by the selective inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo(4,3a)quinoxaline-1-one). Using dynamic contrast enhanced MRI (DCE-MRI) as a minimally invasive technique, we demonstrated for the first time a significant increase in the DCE-MRI read-out initial area under the concentration curve (iAUC60) indicating an acute increase in blood-tumor barrier permeability after i.v. treatment with JS-K. Repeated MR imaging of animals with intracranial U87 gliomas under treatment with JS-K (3.5 μmol/kg JS-K 3×/week) and of untreated controls on day 12 and 19 after tumor inoculation revealed no significant changes in tumor growth, edema formation or tumor perfusion. Immunohistochemical workup of the brains showed a significant antiproliferative effect of JS-K in the gliomas. Taken together, in vitro and in vivo data suggest that JS-K has antiproliferative effects in U87 gliomas and opens the blood-tumor barrier by activation of the NO/cGMP signaling pathway. This might be a novel approach to facilitate entry of therapeutic

Effects of the nitric oxide donor JS-K on the blood-tumor barrier and on orthotopic U87 rat gliomas assessed by MRI.

PubMed

Weidensteiner, Claudia; Reichardt, Wilfried; Shami, Paul J; Saavedra, Joseph E; Keefer, Larry K; Baumer, Brunhilde; Werres, Anna; Jasinski, Robert; Osterberg, Nadja; Weyerbrock, Astrid

2013-04-01

Nitric oxide (NO) released from NO donors can be cytotoxic in tumor cells and can enhance the transport of drugs into brain tumors by altering blood-tumor barrier permeability. The NO donor JS-K [O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] releases NO upon enzymatic activation selectively in cells overexpressing glutathione-S-transferases (GSTs) such as gliomas. Thus, JS-K-dependent NO effects - especially on cell viability and vascular permeability - were investigated in U87 glioma cells in vitro and in an orthotopic U87 xenograft model in vivo by magnetic resonance imaging (MRI). In vitro experiments showed dose-dependent antiproliferative and cytotoxic effects in U87 cells. In addition, treatment of U87 cells with JS-K resulted in a dose-dependent activation of soluble guanylate cyclase and intracellular accumulation of cyclic guanosine monophosphate (cGMP) which was irreversibly inhibited by the selective inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo(4,3a)quinoxaline-1-one). Using dynamic contrast enhanced MRI (DCE-MRI) as a minimally invasive technique, we demonstrated for the first time a significant increase in the DCE-MRI read-out initial area under the concentration curve (iAUC60) indicating an acute increase in blood-tumor barrier permeability after i.v. treatment with JS-K. Repeated MR imaging of animals with intracranial U87 gliomas under treatment with JS-K (3.5 μmol/kg JS-K 3×/week) and of untreated controls on day 12 and 19 after tumor inoculation revealed no significant changes in tumor growth, edema formation or tumor perfusion. Immunohistochemical workup of the brains showed a significant antiproliferative effect of JS-K in the gliomas. Taken together, in vitro and in vivo data suggest that JS-K has antiproliferative effects in U87 gliomas and opens the blood-tumor barrier by activation of the NO/cGMP signaling pathway. This might be a novel approach to facilitate entry of therapeutic

Synthesis, secretion, function, metabolism and application of natriuretic peptides in heart failure.

PubMed

Fu, Shihui; Ping, Ping; Wang, Fengqi; Luo, Leiming

2018-01-01

As a family of hormones with pleiotropic effects, natriuretic peptide (NP) system includes atrial NP (ANP), B-type NP (BNP), C-type NP (CNP), dendroaspis NP and urodilatin, with NP receptor-A (guanylate cyclase-A), NP receptor-B (guanylate cyclase-B) and NP receptor-C (clearance receptor). These peptides are genetically distinct, but structurally and functionally related for regulating circulatory homeostasis in vertebrates. In humans, ANP and BNP are encoded by NP precursor A (NPPA) and NPPB genes on chromosome 1, whereas CNP is encoded by NPPC on chromosome 2. NPs are synthesized and secreted through certain mechanisms by cardiomyocytes, fibroblasts, endotheliocytes, immune cells (neutrophils, T-cells and macrophages) and immature cells (embryonic stem cells, muscle satellite cells and cardiac precursor cells). They are mainly produced by cardiovascular, brain and renal tissues in response to wall stretch and other causes. NPs provide natriuresis, diuresis, vasodilation, antiproliferation, antihypertrophy, antifibrosis and other cardiometabolic protection. NPs represent body's own antihypertensive system, and provide compensatory protection to counterbalance vasoconstrictor-mitogenic-sodium retaining hormones, released by renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS). NPs play central roles in regulation of heart failure (HF), and are inactivated through not only NP receptor-C, but also neutral endopeptidase (NEP), dipeptidyl peptidase-4 and insulin degrading enzyme. Both BNP and N-terminal proBNP are useful biomarkers to not only make the diagnosis and assess the severity of HF, but also guide the therapy and predict the prognosis in patients with HF. Current NP-augmenting strategies include the synthesis of NPs or agonists to increase NP bioactivity and inhibition of NEP to reduce NP breakdown. Nesiritide has been established as an available therapy, and angiotensin receptor blocker NEP inhibitor (ARNI, LCZ696) has obtained

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

Diversity of squalene-hopene cyclases in a tropical carbonate-rich environment

NASA Astrophysics Data System (ADS)

Leavitt, W. D.; Pearson, A.

2007-12-01

Hopanoids are isoprenoid lipids which derive primarily from bacteria and are ubiquitous in contemporary Earth surface environments. In the geologic record, hopanes found in sedimentary rocks are used as proxies to help decipher ancient biological communities. However, in contrast to the ubiquity of these lipid products, biosynthesis of hopanoids appears to be a relatively rare physiological trait among bacteria in complex environmental communities. We have recently estimated that fewer than one in ten bacterial cells in soils and fewer than one in twenty bacterial cells in the ocean contains the gene squalene-hopene cyclase (sqhC) [1]. Biosynthesis of hopanoids is rarer in natural communities than it is among species that have been propagated in pure culture [2]. Here we continue our previous work to survey the phylogeny and diversity of hopanoid producers using culture-independent methods. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analyzed previously [1]. One possible explanation is that hopanoid-producing strains of cyanobacteria are regionally localized. It has been suggested that throughout the long-term sedimentary record there is a correlation between 2-methylhopanoid index (a putative indicator of cyanobacterial biomass) and the global prevalence of shallow carbonate platform environments [3], and in previous work we did not analyze any such environments. To address this question we surveyed a land-sea gradient across the Bahamian island of San Salvador. Samples were taken from upland soil, a hypersaline lake, a tidal creek, and the shallow open ocean. The data are remarkably similar to our previous results: environmental sqhCs average < 65% translated amino acid identity to their closest relatives in public databases, and non- cyanobacterial sequences continue to dominate. We will discuss the challenges these results pose for deciphering the global distribution of microbially

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

PubMed Central

Rismondo, Jeanine; Gibhardt, Johannes; Rosenberg, Jonathan; Kaever, Volkhard

2015-01-01

ABSTRACT Cyclic diadenylate monophosphate (c-di-AMP) is a second messenger utilized by diverse bacteria. In many species, including the Gram-positive human pathogen Listeria monocytogenes, c-di-AMP is essential for growth. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with its potential regulatory protein, CdaR, via the transmembrane protein domain. The presence of the CdaR protein is not required for the membrane localization and abundance of CdaA. We have also found that CdaR negatively influences CdaA activity in L. monocytogenes and that the role of CdaR is most evident at a high growth temperature. Interestingly, a cdaR mutant strain is less susceptible to lysozyme. Moreover, CdaA contributes to cell division, and cells depleted of CdaA are prone to lysis. The observation that the growth defect of a CdaA depletion strain can be partially restored by increasing the osmolarity of the growth medium suggests that c-di-AMP is important for maintaining the integrity of the protective cell envelope. Overall, this work provides new insights into the relationship between CdaA and CdaR. IMPORTANCE Cyclic diadenylate monophosphate (c-di-AMP) is a recently identified second messenger that is utilized by the Gram-positive human pathogen Listeria monocytogenes. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with CdaR, its potential regulatory protein. We show that CdaR is not required for membrane localization or abundance of the diadenylate cyclase, but modulates its activity. Moreover, CdaA seems to contribute to cell division. Overall, this work provides new insights into the relationship between CdaA and CdaR and their involvement in cell growth. PMID:26527648

AN ADENYLYL CYCLASE SIGNALING PATHWAY PREDICTS DIRECT DOPAMINERGIC INPUT TO VESTIBULAR HAIR CELLS

PubMed Central

DRESCHER, M. J.; CHO, W. J.; FOLBE, A. J.; SELVAKUMAR, D.; KEWSON, D. T.; ABU-HAMDAN, M. D.; OH, C. K.; RAMAKRISHNAN, N. A.; HATFIELD, J. S.; KHAN, K. M.; ANNE, S.; HARPOOL, E. C.; DRESCHER, D. G.

2010-01-01

Adenylyl cyclase signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across adenylyl cyclase (AC) isoforms, and RT-PCR, coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca2+, the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca2+. Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate adenylyl cyclase activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. While a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC

Current and Novel Therapeutic Options for Irritable Bowel Syndrome Management

PubMed Central

Camilleri, Michael; Andresen, Viola

2009-01-01

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder affecting up to 3-15% of the general population in western countries. It is characterized by unexplained abdominal pain, discomfort, and bloating in association with altered bowel habits. The pathophysiology of IBS is multifactorial involving disturbances of the brain-gut-axis. The pathophysiology provides the rationale for pharmacotherapy: abnormal gastrointestinal motor functions, visceral hypersensitivity, psychosocial factors, autonomic dysfunction, and mucosal immune activation. Understanding the mechanisms, and their mediators or modulators including neurotransmitters and receptors have led to several therapeutic approaches including agents acting on the serotonin receptor or serotonin transporter system, antidepressants, novel selective anticholinergics, α-adrenergic agonists, opioid agents, cholecystokinin-antagonists, neurokinin-antagonists, somatostatin receptor agonists, corticotropin releasing factor antagonists, chloride-channel activators, guanylate-cyclase-c agonists, melatonin, atypical benzodiazepines, antibiotics, immune modulators and probiotics. The mechanisms and current evidence regarding efficacy of these agents are reviewed. PMID:19665953

Aminoethyl nitrate – the novel super nitrate?

PubMed Central

Bauersachs, Johann

2009-01-01

Long-term use of most organic nitrates is limited by development of tolerance, induction of oxidative stress and endothelial dysfunction. In this issue of the BJP, Schuhmacher et al. characterized a novel class of organic nitrates with amino moieties (aminoalkyl nitrates). Aminoethyl nitrate was identified as a novel organic mononitrate with high potency but devoid of induction of mitochondrial oxidative stress. Cross-tolerance to nitroglycerin or the endothelium-dependent agonist acetylcholine after in vivo treatment was not observed. Like all nitrates, aminoethyl nitrate induced vasorelaxation by activation of soluble guanylate cyclase. Thus, in contrast to the prevailing view, high potency in an organic nitrate is not necessarily accompanied by induction of oxidative stress or endothelial dysfunction. This work from Daiber's group is an important step forward in the understanding of nitrate bioactivation, tolerance phenomena and towards the development of better organic nitrates for clinical use. PMID:19732062

Radiolabeled Escherichia coli heat-stable enterotoxin analogs for in vivo imaging of colorectal cancer

NASA Astrophysics Data System (ADS)

Giblin, M. F.; Sieckman, G. L.; Owen, N. K.; Hoffman, T. J.; Forte, L. R.; Volkert, W. A.

2005-12-01

The human Escherichia coli heat-stable enterotoxin (STh, amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. In the current study, two STh analogs were synthesized and evaluated in vitro and in vivo. Both analogs shared identical 6-19 core sequences, and had N-terminal pendant DOTA moieties. The analogs differed in the identity of a 6 amino acid peptide sequence intervening between DOTA and the 6-19 core. In one analog, the peptide was an RGD-containing sequence found in human fibronectin (GRGDSP), while in the other this peptide sequence was randomly scrambled (GRDSGP). The results indicated that the presence of the human fibronectin sequence in the hybrid peptide did not affect tumor localization in vivo.

Characterization, Purification of Poncirin from Edible Citrus Ougan (Citrus reticulate cv. Suavissima) and Its Growth Inhibitory Effect on Human Gastric Cancer Cells SGC-7901

PubMed Central

Zhu, Xiaoyan; Luo, Fenglei; Zheng, Yixiong; Zhang, Jiukai; Huang, Jianzhen; Sun, Chongde; Li, Xian; Chen, Kunsong

2013-01-01

Poncirin is a bitter flavanone glycoside with various biological activities. Poncirin was isolated from four different tissues (flavedo, albedo, segment membrane, and juice sac) of Ougan fruit (Citrus reticulate cv. Suavissima). The highest content of poncirin was found in the albedo of Ougan fruit (1.37 mg/g DW). High speed counter-current chromatography (HSCCC) combined with D101 resin chromatography was utilized for the separation and purification of poncirin from the albedo of Ougan fruit. After this two-step purification, poncirin purity increased from 0.14% to 96.56%. The chemical structure of the purified poncirin was identified by both HPLC-PDA and LC-MS. Poncirin showed a significant in vitro inhibitory effect on the growth of the human gastric cancer cells, SGC-7901, in a dose-dependent manner. Thus, poncirin from Ougan fruit, may be beneficial for gastric cancer prevention. The purification method demonstrated here will be useful for further studies on the pharmacological mechanism of poncirin activity, as well as for guiding the consumption of Ougan fruit. PMID:23615464

An adenylyl cyclase gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

USDA-ARS?s Scientific Manuscript database

The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in som...

Synthesis and characterization of novel ion-imprinted guanyl-modified cellulose for selective extraction of copper ions from geological and municipality sample.

PubMed

Kenawy, I M; Ismail, M A; Hafez, M A H; Hashem, M A

2018-04-21

The new ion-imprinted guanyl-modified cellulose (II.Gu-MC) was prepared for the separation and determination of Cu (II) ions in different real samples. Several techniques such as Fourier Transform Infrared (FT-IR), scanning electron microscope (SEM), thermal analysis, potentiograph and elemental analysis have been utilized for the characterization of II.Gu-MC. The adsorption behavior of the ion imprinted polymer (II.Gu-MC) was evaluated and compared to the non ion-imprinted polymer (NII.Gu-MC) at the optimum conditions. The selectivity and the adsorption capacity were greatly enhanced by using the ion-imprinted polymer, indicating its validation for the separation and determination of Cu 2+ ions in different matrices. The adsorption capacity by chelating fibers II.Gu-MC & NII.Gu-MC agreed with the second-order model, and the sorption-isotherm experiments revealed best agreement with Langmuir model. The adsorption capacity of II.Gu-MC and NII.Gu-MC were 115 and 55 mg·g -1 , respectively. The II.Gu-MC was successfully employed for the selective separation and determination of Cu(II) ions with high accuracy. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure of RNA 3′-phosphate cyclase bound to substrate RNA

PubMed Central

Desai, Kevin K.; Bingman, Craig A.; Cheng, Chin L.; Phillips, George N.

2014-01-01

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 Nε 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. PMID:25161314

Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase.

PubMed

Venkataramani, Prabhadevi; Liang, Zhao-Xun

2017-01-01

C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs. We have identified and engineered a thermophilic DGC for efficient production of c-di-GMP for characterizing c-di-GMP signaling proteins and riboswitches. Importantly, residue replacement in the inhibitory I-site of the thermophilic DGC drastically relieved product inhibition to enable the production of hundreds of milligrams of c-di-GMP using 5-10 mg of this robust biocatalyst.

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.

PubMed

Kühnel, Lisa C; Nestl, Bettina M; Hauer, Bernhard

2017-11-16

Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

PubMed

Saier, M H; Keeler, D K; Feucht, B U

1982-03-10

Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

PubMed

Abergel, Chantal; Blanc, Guillaume; Monchois, Vincent; Renesto, Patricia; Sigoillot, Cécile; Ogata, Hiroyuki; Raoult, Didier; Claverie, Jean-Michel

2006-11-01

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Role of nitric oxide and cyclic GMP signaling in melanocyte response to hypergravity

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Lambers, Britta; Tsiockas, Wasiliki; Block, Ingrid; Gerzer, Rupert

Nitric oxide (NO) has a prominent role in many (patho)physiological processes in the skin including erythema, inflammation, and cancerogenesis. The soluble guanylyl cyclase (sGC), a key transducer in NO signaling, catalyzes the formation of the second messenger guanosine 3´,5´-cyclic monophosphate (cyclic cGMP or cGMP). For human melanocytes, which are responsible for skin pigmentation by synthesizing the pigment melanin, it has been reported that the NO/sGC/cGMP pathway is involved in UVB-induced melanogenesis. Melanin acts as a scavenger for free radicals that may arise during metabolic stress. It may also act as a photosensitizer that generates active oxygen species upon UV irradiation, which may initiate hypopigmentary disorders (e.g., vitiligo) as well as UV-induced oncogene cell transformation. In addition, melanoma, a deadly skin cancer, which arises from transformed melanocytes, is characterized by a resistance to chemotherapy. In our studies we have shown that NO can induce perturbation of melanocyte-extracellular matrix component interactions, which may contribute to loss of melanocytes or melanoma metastasis. Such NO effects appear to be modulated partly via cGMP. Moreover, we found that different guanylyl cyclase isoforms are responsible for cGMP synthesis in melanocytic cells. Normal human melanocytes and nonmetastatic melanoma cells predominantly express sGC, which appears to be associated with melanogenesis, whereas absence of NO-sensitive GC, but up-regulated activities of the natriuretic peptide-sensitive membrane guanylyl cyclase isoforms were found in highly metastatic phenotypes. Due to the growing interest in the regulation of signaling activities in normal and transformed cells under altered gravity conditions, we have further investigated whether the NO/cGMP signaling is involved in melanocyte response to gravitational stress. We found that normal human melanocytes and non-metastatic melanoma cell lines, but not highly metastatic cells

cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)β(1).

PubMed

Beste, Kerstin Y; Seifert, Roland

2013-02-01

Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

PubMed Central

2010-01-01

Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

PubMed Central

Modiano, Nir; Lu, Yanping E.; Cresswell, Peter

2005-01-01

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-γ-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-γ. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result. PMID:15937107

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.

PubMed

Modiano, Nir; Lu, Yanping E; Cresswell, Peter

2005-06-14

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.

The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

PubMed

Ramírez-Mata, Alberto; López-Lara, Lilia I; Xiqui-Vázquez, Ma Luisa; Jijón-Moreno, Saúl; Romero-Osorio, Angelica; Baca, Beatriz E

2016-04-01

In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

PubMed

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The impact of nitric oxide in cardiovascular medicine: untapped potential utility.

PubMed

Pepine, Carl J

2009-05-01

The structural integrity and functional activity of the endothelium play an important role in atherogenesis and related adverse outcomes. Cardiovascular disease risk conditions contribute to oxidative stress, which causes a disruption in the balance between nitric oxide (NO) and reactive oxygen species, with a resulting relative decrease in bioavailable NO and/or the NO-soluble guanylate cyclase cascade in blood vessels. This leads to endothelial and vascular smooth muscle cell dysfunction, resulting in increased tone and alterations in cell growth and gene expression that create a prothrombotic, proinflammatory environment. This leads to formation, progression, and destabilization of atherosclerotic plaques which may result in myocardial infarction, stroke, and cardiovascular death. NO clearly has a critical role in the maintenance and repair of the vasculature, and a decrease in bioavailable NO is linked to adverse outcomes. This background provides the rationale for exploring the potential therapeutic role for NO-donating agents in the prevention of adverse cardiovascular outcomes.

Antioxidant and Vasodilator Activity of Ugni molinae Turcz. (Murtilla) and Its Modulatory Mechanism in Hypotensive Response

PubMed Central

Jofré, Ignacio; Pezoa, Cesar; Scheuermann, Erick; Rosalen, Pedro Luiz; Romero, Fernando

2016-01-01

Hypertension is a systemic condition with high morbidity and mortality rates worldwide, which poses an increased risk for cardiovascular diseases. In this study, we demonstrated the antioxidant and vasodilator activity of Ugni molinae Turcz. (Murtilla) fruit, a berry native to Chile and proposed models to explain its modulatory mechanism in hypotensive response. Murtilla fruits were cultivated in a germplasm bank and submitted to chemical and biological analyses. The phenolic compounds gallic acid, Catechin, Quercetin-3-β-D-glucoside, Myricetin, Quercetin, and Kaempferol were identified. Murtilla extract did not generate toxic effects on human endothelial cells and had significant antioxidant activity against ROS production, lipid peroxidation, and superoxide anion production. Furthermore, it showed dose-dependent vasodilator activity in aortic rings in the presence of endothelium, whose hypotensive mechanism is partially mediated by nitric oxide synthase/guanylate cyclase and large-conductance calcium-dependent potassium channels. Murtilla fruits might potentially have beneficial effects on the management of cardiovascular diseases. PMID:27688827

New treatment options for chronic constipation: Mechanisms, efficacy and safety

PubMed Central

Camilleri, Michael

2011-01-01

The present review has several objectives, the first of which is to review the pharmacology and selectivity of serotonergic agents to contrast the older serotonergic agents (which were withdrawn because of cardiac or vascular adverse effects) with the newer generation serotonin receptor subtype 4 agonists. Second, the chloride ion secretagogues that act through the guanylate cyclase C receptor are appraised and their pharmacology is compared with the approved medication, lubiprostone. Third, the efficacy and safety of the application of bile acid modulation to treat constipation are addressed. The long-term studies of surgically induced excess bile acid delivery to the colon are reviewed to ascertain the safety of this therapeutic approach. Finally, the new drugs for opiate-induced constipation are introduced. Assuming these drugs are approved, practitioners will have a choice; however, patient responsiveness will be based on trial and error. Nevertheless, the spectrum of mechanisms and demonstrated efficacy and safety augur well for satisfactory treatment outcomes. PMID:22114755

Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rybin, V.O.; Gureeva, A.A.

1986-05-10

The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature ofmore » the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.« less

Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

PubMed Central

Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

2012-01-01

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

New Therapeutic Agent against Arterial Thrombosis: An Iridium(III)-Derived Organometallic Compound.

PubMed

Hsia, Chih-Wei; Velusamy, Marappan; Tsao, Jeng-Ting; Hsia, Chih-Hsuan; Chou, Duen-Suey; Jayakumar, Thanasekaran; Lee, Lin-Wen; Li, Jiun-Yi; Sheu, Joen-Rong

2017-12-05

Platelet activation plays a major role in cardio and cerebrovascular diseases, and cancer progression. Disruption of platelet activation represents an attractive therapeutic target for reducing the bidirectional cross talk between platelets and tumor cells. Platinum (Pt) compounds have been used for treating cancer. Hence, replacing Pt with iridium (Ir) is considered a potential alternative. We recently developed an Ir(III)-derived complex, [Ir(Cp*)1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine Cl]BF₄ (Ir-11), which exhibited strong antiplatelet activity; hence, we assessed the therapeutic potential of Ir-11 against arterial thrombosis. In collagen-activated platelets, Ir-11 inhibited platelet aggregation, adenosine triphosphate (ATP) release, intracellular Ca 2+ mobilization, P-selectin expression, and OH · formation, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt. Neither the adenylate cyclase inhibitor nor the guanylate cyclase inhibitor reversed the Ir-11-mediated antiplatelet effects. In experimental mice, Ir-11 prolonged the bleeding time and reduced mortality associated with acute pulmonary thromboembolism. Ir-11 plays a crucial role by inhibiting platelet activation through the inhibition of the PLCγ2-PKC cascade, and the subsequent suppression of Akt and MAPK activation, ultimately inhibiting platelet aggregation. Therefore, Ir-11 can be considered a new therapeutic agent against either arterial thrombosis or the bidirectional cross talk between platelets and tumor cells.

Disruption of type 3 adenylyl cyclase expression in the hypothalamus leads to obesity

PubMed Central

Cao, Hong; Chen, Xuanmao; Yang, Yimei; Storm, Daniel R

2016-01-01

Evidence from human studies and transgenic mice lacking the type 3 adenylyl cyclase (AC3) indicates that AC3 plays a role in the regulation of body weight. It is unknown in which brain region AC3 exerts such an effect. We examined the role of AC3 in the hypothalamus for body weight control using a floxed AC3 mouse strain. Here, we report that AC3 flox/flox mice became obese after the administration of AAV-CRE-GFP into the hypothalamus. Both male and female AC3 floxed mice showed heavier body weight than AAV-GFP injected control mice. Furthermore, mice with selective ablation of AC3 expression in the ventromedial hypothalamus also showed increased body weight and food consumption. Our results indicated that AC3 in the hypothalamus regulates energy balance. PMID:27942392

The impact of long-term systemic glucocorticoid use in severe asthma: A UK retrospective cohort analysis.

PubMed

Daugherty, Jonas; Lin, Xiwu; Baxter, Richard; Suruki, Robert; Bradford, Eric

2018-06-01

Systemic glucocorticoids (SGCs) are a treatment option for severe asthma but are associated with the development of adverse events (AEs). Evidence on the extent of SGC use and the relationship between SGC dose and AE risk in severe asthma is limited. Patients with severe asthma (Global Initiative for Asthma step 4/5), with no SGC use during the <6-12 months before severe asthma determination (index date) were identified in the UK-based Clinical Practice Research Datalink database (2004-2012). Patients were assessed for SGC exposure and an incident diagnosis of an SGC-related AE (cataracts, diabetes, myocardial infarction [MI], osteoporosis, peptic ulcer or stroke) during the 8-year observation phase. The dose-related risk of an SGC-related AE was determined using AE-specific Cox proportional hazards models. Overall, 75% of 60,418 patients identified with severe asthma received SGC during the 8-year follow-up, with the majority receiving an average of >0-≤2.5 mg/day. The risk of diabetes (hazard ratio [HR]:1.20 [95% confidence interval (CI): 1.11, 1.30]), MI (HR: 1.25 [95% CI: 1.09, 1.43]) and osteoporosis (HR: 1.64 [95% CI: 1.51, 1.78]) was increased at low SGC doses (0-2.5 mg/day), with further risk increases at doses >2.5 mg/day versus no SGC use. Compared with no SGC use, SGC increased the risk of peptic ulcer in a non-dose-dependent manner, but the risk of stroke was unchanged. Most patients with severe asthma are exposed to SGC, which increases SGC-related AE risk. This suggests that SGC exposure should be minimized as recommended by asthma treatment guidelines.

Genetic alterations in the NO-cGMP pathway and cardiovascular risk.

PubMed

Wobst, Jana; Schunkert, Heribert; Kessler, Thorsten

2018-06-01

In the past ten years, several chromosomal loci have been identified by genome-wide association studies to influence the risk of coronary artery disease (CAD) and its risk factors. The GUCY1A3 gene encoding the α 1 subunit of the soluble guanylyl cyclase (sGC) resides at one of these loci and has been strongly associated with blood pressure and CAD risk. More recently, further genes in the pathway encoding the endothelial nitric oxide synthase, the phosphodiesterases 3A and 5A, and the inositol 1,4,5-trisphosphate receptor I-associated protein (IRAG), i.e., NOS3, PDE3A, PDE5A, and MRVI1, respectively, were likewise identified as CAD risk genes. In this review, we highlight the genetic findings linking variants in NO-cGMP signaling and cardiovascular disease, discuss the potential underlying mechanisms which might propagate the development of atherosclerosis, and speculate about therapeutic implications. Copyright © 2018 Elsevier Inc. All rights reserved.

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

PubMed Central

Emala, C. W.; Aryana, A.; Hirshman, C. A.

1996-01-01

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

PubMed Central

Shuhaibar, Leia C.; Egbert, Jeremy R.; Edmund, Aaron B.; Uliasz, Tracy F.; Dickey, Deborah M.; Yee, Siu-Pok; Potter, Lincoln R.; Jaffe, Laurinda A.

2016-01-01

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events. PMID:26522847

Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.

PubMed

Choi, Hyun B; Gordon, Grant R J; Zhou, Ning; Tai, Chao; Rungta, Ravi L; Martinez, Jennifer; Milner, Teresa A; Ryu, Jae K; McLarnon, James G; Tresguerres, Martin; Levin, Lonny R; Buck, Jochen; MacVicar, Brian A

2012-09-20

Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

PubMed Central

Choi, Hyun B.; Gordon, Grant R.J.; Zhou, Ning; Tai, Chao; Rungta, Ravi L.; Martinez, Jennifer; Milner, Teresa A.; Ryu, Jae K.; McLarnon, James G.; Tresguerres, Martin; Levin, Lonny R.; Buck, Jochen; MacVicar, Brian A.

2013-01-01

SUMMARY Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO3−) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO3− entry via the electrogenic NaHCO3 cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K+]ext and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. PMID:22998876

Transmembrane adenylyl cyclase regulates amphibian sperm motility through Protein Kinase A activation

PubMed Central

O’Brien, Emma D.; Krapf, Darío; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

2014-01-01

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation. PMID:21126515

Inhibitors for human glutaminyl cyclase by structure based design and bioisosteric replacement.

PubMed

Buchholz, Mirko; Hamann, Antje; Aust, Susanne; Brandt, Wolfgang; Böhme, Livia; Hoffmann, Torsten; Schilling, Stephan; Demuth, Hans-Ulrich; Heiser, Ulrich

2009-11-26

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

PubMed

Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

2018-03-28

G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

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. Copyright © 2016 the American Physiological Society.

Hydrogen Sulfide Regulates Ca2+ Homeostasis Mediated by Concomitantly Produced Nitric Oxide via a Novel Synergistic Pathway in Exocrine Pancreas

PubMed Central

Moustafa, Amira

2014-01-01

Abstract Aim: The present study was designed to explore the effects of hydrogen sulfide (H2S) on Ca2+ homeostasis in rat pancreatic acini. Results: Sodium hydrosulfide (NaHS; an H2S donor) induced a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The NaHS-induced [Ca2+]i elevation persisted with an EC50 of 73.3 μM in the absence of extracellular Ca2+ but was abolished by thapsigargin, indicating that both Ca2+ entry and Ca2+ release contributed to the increase. The [Ca2+]i increase was markedly inhibited in the presence of NG-monomethyl L-arginine or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and diaminofluorescein-2/diaminofluorescein-2 triazole (DAF-2/DAF-2T) fluorometry demonstrated that nitric oxide (NO) was also produced by H2S in a dose-dependent manner with an EC50 of 64.8 μM, indicating that NO was involved in the H2S effect. The H2S-induced [Ca2+]i increase was inhibited by pretreatment with U73122, xestospongin C, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, KT5823, and GP2A, indicating that phospholipase C (PLC), the inositol 1,4,5-trisphosphate (IP3) receptor, soluble guanylate cyclase (sGC), protein kinase G (PKG), and Gq-protein play roles as intermediate components in the H2S-triggered intracellular signaling. Innovation: To our knowledge, our study is the first one highlighting the effect of H2S on intracellular Ca2+ dynamics in pancreatic acinar cells. Moreover, a novel cascade was presumed to function via the synergistic interaction between H2S and NO. Conclusion: We conclude that H2S affects [Ca2+]i homeostasis that is mediated by H2S-evoked NO production via an endothelial nitric oxide synthase (eNOS)-NO-sGC-cyclic guanosine monophosphate-PKG-Gq-protein-PLC-IP3 pathway to induce Ca2+ release, and this pathway is identical to the one we recently proposed for a sole effect of NO and the two gaseous molecules synergistically function to regulate Ca2+ homeostasis

Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

PubMed

Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

2010-11-01

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Preclinical evaluation of collagen type I scaffolds, including gelatin-collagen microparticles and loaded with a hydroglycolic Calendula officinalis extract in a lagomorph model of full-thickness skin wound.

PubMed

Millán, D; Jiménez, R A; Nieto, L E; Linero, I; Laverde, M; Fontanilla, M R

2016-02-01

Previously, we have developed collagen type I scaffolds including microparticles of gelatin-collagen type I (SGC) that are able to control the release of a hydroglycolic extract of the Calendula officinalis flower. The main goal of the present work was to carry out the preclinical evaluation of SGC alone or loaded with the C. officinalis extract (SGC-E) in a lagomorph model of full-thickness skin wound. A total of 39 rabbits were distributed in three groups, of 13 animals each. The first group was used to compare wound healing by secondary intention (control) with wound healing observed when wounds were grafted with SGC alone. Comparison of control wounds with wounds grafted with SGC-E was performed in the second group, and comparison of wounds grafted with SGC with wounds grafted with SGC-E was performed in the third group. Clinical follow-ups were carried in all animals after surgery, and histological and histomorphometric analyses were performed on tissues taken from the healed area and healthy surrounding tissue. Histological and histomorphometric results indicate that grafting of SGC alone favors wound healing and brings a better clinical outcome than grafting SGC-E. In vitro collagenase digestion data suggested that the association of the C. officinalis extract to SGC increased the SGC-E cross-linking, making it difficult to degrade and affecting its biocompatibility.

The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

PubMed Central

Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

2009-01-01

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.

PubMed

Penzkofer, Alfons; Stierl, Manuela; Mathes, Tilo; Hegemann, Peter

2014-11-01

The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure-activity relationships of benzimidazole-based glutaminyl cyclase inhibitors featuring a heteroaryl scaffold.

PubMed

Ramsbeck, Daniel; Buchholz, Mirko; Koch, Birgit; Böhme, Livia; Hoffmann, Torsten; Demuth, Hans-Ulrich; Heiser, Ulrich

2013-09-12

Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of Alzheimer's disease (AD). The inhibition of hQC prevents of the formation of the Aβ3(pE)-40,42 species which were shown to be of elevated neurotoxicity and are likely to act as a seeding core, leading to an accelerated formation of Aβ-oligomers and fibrils. This work presents a new class of inhibitors of hQC, resulting from a pharmacophore-based screen. Hit molecules were identified, containing benzimidazole as the metal binding group connected to 1,3,4-oxadiazole as the central scaffold. The subsequent optimization resulted in benzimidazolyl-1,3,4-thiadiazoles and -1,2,3-triazoles with an inhibitory potency in the nanomolar range. Further investigation into the potential binding mode of the new compound classes combined molecular docking and site directed mutagenesis studies.

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, T.; Ui, M.

1985-06-25

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

Mechanisms involved in the relaxant action of the ethanolic extract of propolis in the guinea-pig trachea in-vitro.

PubMed

Paulino, Niraldo; Scremin, Fernando M; Raichaski, Lisiane B; Marcucci, Maria Cristina; Scremin, Amarilis; Calixto, João B

2002-06-01

This study examines the mechanisms by which the standardised ethanolic extract of propolis induces relaxation of the guinea-pig trachea in-vitro. In guinea-pig trachea with or without epithelium and contracted by histamine, the propolis extract caused reproducible and graded relaxation, with a mean EC50 value of 3.8 or 10.5 microg mL(-1) and Emax of 100%, respectively. The propolis extract-induced relaxation was markedly reduced (26+/-9 and 96+/-3%) when guinea-pig tracheas were exposed to Krebs solution containing elevated K+ in the medium (40 or 80 mM). Pre-incubation of guinea-pig tracheas with tetraethylamonium (100 mM) or with 4-aminopyridine (10mM) reduced the propolis extract-induced relaxation by 31+/-10% and 28+/-2%. Likewise, apamin (0.1 microM), charybdotoxin (0.1 microM) or iberiotoxin (0.1 microM) caused marked inhibition of propolis extract-mediated relaxation in guinea-pig trachea (percentage of inhibition: 65+/-3%, 60+/-5% and 65+/-9%, respectively). Also, glibenclamide (1 microM) inhibited the relaxant response caused by the propolis extract by 57+/-4%. Omega-conotoxin GIVA (0.1 microM) or capsaicin (1 microM) produced small but significant inhibition (30+/-5% or 47+/-7%, respectively) of the propolis extract-induced relaxation. The vasoactive intestinal peptide (VIP) antagonist D-p-Cl-Phe6,Leu17[VIP] porcine (0.1 microM) inhibited relaxation by 55+/-5%, while propranolol (1 microM) induced a parallel rightward displacement (about 20 fold) of the propolis extract concentration-response curve. Finally, the propolis extract-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-N(G)-nitroarginine (L-NOArg, 100 microM) (48+/-6%), and by the soluble guanylatecyclase inhibitormethylene blue (10 microM) (37+/-6%), whilethe moreselectivesoluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3-alquinoxalin-1-one (ODQ, 1 microM) produced only a parallel (about 3 fold) rightward displacement of the propolis extract concentration

Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slotkin, T.A.; Navarro, H.A.; McCook, E.C.

1990-01-01

Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group.more » These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

PubMed

Hess, Kenneth C; Jones, Brian H; Marquez, Becky; Chen, Yanqiu; Ord, Teri S; Kamenetsky, Margarita; Miyamoto, Catarina; Zippin, Jonathan H; Kopf, Gregory S; Suarez, Susan S; Levin, Lonny R; Williams, Carmen J; Buck, Jochen; Moss, Stuart B

2005-08-01

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, S.B.; Halenda, S.P.; Bylund, D.B.

1991-02-01

The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipasemore » A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.« less

Chronic treatment with escitalopram but not R-citalopram translocates Galpha(s) from lipid raft domains and potentiates adenylyl cyclase: a 5-hydroxytryptamine transporter-independent action of this antidepressant compound.

PubMed

Zhang, Lanqiu; Rasenick, Mark M

2010-03-01

Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galpha(s) from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Galpha(s) in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Galpha(s) in lipid rafts, whereas there was no change in overall Galpha(s) content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Galpha(s) localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Galpha(s) and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Galpha(s) from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.

Chronic Treatment with Escitalopram but Not R-Citalopram Translocates Gαs from Lipid Raft Domains and Potentiates Adenylyl Cyclase: A 5-Hydroxytryptamine Transporter-Independent Action of This Antidepressant Compound

PubMed Central

Zhang, Lanqiu

2010-01-01

Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Gαs from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Gαs in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Gαs in lipid rafts, whereas there was no change in overall Gαs content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Gαs localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Gαs and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Gαs from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs. PMID:19996298

Diversity in Guanosine 3′,5′-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants*

PubMed Central

Nomura, Yuhta; Izumi, Atsushi; Fukunaga, Yoshinori; Kusumi, Kensuke; Iba, Koh; Watanabe, Seiya; Nakahira, Yoichi; Weber, Andreas P. M.; Nozawa, Akira; Tozawa, Yuzuru

2014-01-01

The guanosine 3′,5′-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μm relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μm. The IC50 of ppGpp for GKpm was ∼10 μm. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp. PMID:24722991

Synechocystis sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll a for activity.

PubMed

Xiong, Wei; Shen, Gaozhong; Bryant, Donald A

2017-03-01

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA 6803 ) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA 6803 gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA 6803 was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA 6803 was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA 6803 is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.

Interference of P-REX2a may inhibit proliferation and reverse the resistance of SGC7901 cells to doxorubicin.

PubMed

Ai, Yaowei; Zhou, Qiaohui; Li, Ling; Pan, Zhihong; Guo, Mingwen; Han, Jingbo

2018-03-01

Drug resistance inhibits the efficacy of doxorubicin in gastric cancer. Phosphatidylinositol 3,4,5-trisphosphate RAC exchanger 2a (P-REX2a) activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway by binding to and inactivating phosphatase and tensin homolog (PTEN), which functions as a tumor promoter in a number of types of cancer. However, there is no research concerning the association between P-REX2a expression and drug resistance in gastric cancer. In the present study, the expression of P-REX2a in clinical gastric cancer tissues was detected, and the mechanism of doxorubicin resistance in the gastric cancer cell line SGC7901 was investigated. Using reverse transcription-quantitative polymerase chain reaction and western blotting, it was demonstrated that the mRNA and protein expression of P-REX2a was increased in gastric cancer tissues. MTT assays were also used to determine proliferation, and proliferation was revealed to be reduced following transfection of P-REX2a small interfering (si)RNA. When the cells were treated with 0.3 µM doxorubicin for 24 h, the rate of apoptosis in the siRNA-transfected groups significantly increased and no marked changes in of PTEN and Akt expression were observed. By contrast, the activity of PTEN increased, and the expression of p-Akt (S473) decreased in the P-REX2a siRNA-transfected group compared with the control. The detection of PTEN enzymatic activity in the present study was based on phosphatidylinositol-3,4,5-trisphosphate. Therefore, it was concluded that P-REX2a may participate in the generation of resistance to doxorubicin in gastric cancer, and this may be associated with the upregulation of the PI3K/Akt signaling pathway via inactivation of PTEN.

California Space Grant Consortium

NASA Technical Reports Server (NTRS)

Kosmatka, John; Berger, Wolfgang; Wiskerchen, Michael J.

2005-01-01

The organizational and administrative structure of the CaSGC has the Consortium Headquarters Office (Principal Investigator - Dr. John Kosmatka, California Statewide Director - Dr. Michael Wiskerchen) at UC San Diego. Each affiliate member institution has a campus director and an scholarship/fellowship selection committee. Each affiliate campus director also serves on the CaSGC Advisory Council and coordinates CMIS data collection and submission. The CaSGC strives to maintain a balance between expanded affiliate membership and continued high quality in targeted program areas of aerospace research, education, workforce development, and public outreach. Associate members are encouraged to participate on a project-by-project basis that meets the needs of California and the goals and objectives of the CaSGC. Associate members have responsibilities relating only to the CaSGC projects they are directly engaged in. Each year, as part of the CaSGC Improvement Plan, the CaSGC Advisory Council evaluates the performance of the affiliate and associate membership in terms of contributions to the CaSGC Strategic Plan, These CaSGC membership evaluations provide a constructive means for elevating productive members and removing non-performing members. This Program Improvement and Results (PIR) report will document CaSGC program improvement results and impacts that directly respond to the specific needs of California in the area of aerospace-related education and human capital development and the Congressional mandate to "increase the understanding, assessment, development and utilization of space resources by promoting a strong education base, responsive research and training activities, and broad and prompt dissemination of knowledge and technology".

Reduced sensitivity of the hepatic adenylate cyclase-cyclic AMP system to glucagon during sustained hormonal stimulation.

PubMed Central

DeRubertis, F R; Craven, P

1976-01-01

Hormone-induced desensitization of hormonal regulation of cyclic AMP (cAMP) content has been described in a number of tissues. In the present study, we examined responses of rat liver to glucagon after periods of sustained exposure to the hormone in vivo and in vitro. In intact anesthetized rats infused with glucagon (50 ng/min) for 1 h or more and in liver slices incubated with the hormone (10 muM) for this period, hepatic cAMP responsiveness to glucagon was significantly blunted compared with that of tissue exposed to the hormone for shorter periods. The reduction in hepatic cAMP responsiveness to glucagon appeared to be fully expressed by 2 h. With the doses of hormone employed, the sequential alterations in hepatic responsiveness seemed to be limited to the cAMP system, since other parameters of glucagon action did not wane with time. Diminished hepatic cAMP responsiveness during sustained hormonal exposure could not be attributed to decreased glucagon availability, accelerated extracellular release of cAMP, hepatic ATP depletion, or enhanced phosphodiesterase activity. Studies in vitro suggested that modulation of the cAMP response occurred at the level of adenylate cyclase (AC). During sustained exposure of hepatic slices to glucagon, reductions in glucagon-responsive AC correlated temporally with those in cAMP and both changes were reversible. Alterations in glucagon-responsive AC were demonstrated over a wide range of ATP (10 muM-0.1 mM) and glucagon (10 nM-5 MM) concentrations in the cyclase reaction mixture, and appeared to be a noncompetitive phenomenon relative to glucagon. Maximal NaF-responsive AC did not fall concomitantly with time. Thus, the reduction in glucagon-responsive AC was probably not related to a reduction in the catalytic unit of the enzyme, but could have been due to an alteration in glucagon binding to its receptor sites, or in the coupling mechanism involved in transmission of the hormonal signal to the catalytic unit. Images PMID

GCPred: a web tool for guanylyl cyclase functional centre prediction from amino acid sequence.

PubMed

Xu, Nuo; Fu, Dongfang; Li, Shiang; Wang, Yuxuan; Wong, Aloysius

2018-06-15

GCPred is a webserver for the prediction of guanylyl cyclase (GC) functional centres from amino acid sequence. GCs are enzymes that generate the signalling molecule cyclic guanosine 3', 5'-monophosphate from guanosine-5'-triphosphate. A novel class of GC centres (GCCs) has been identified in complex plant proteins. Using currently available experimental data, GCPred is created to automate and facilitate the identification of similar GCCs. The server features GCC values that consider in its calculation, the physicochemical properties of amino acids constituting the GCC and the conserved amino acids within the centre. From user input amino acid sequence, the server returns a table of GCC values and graphs depicting deviations from mean values. The utility of this server is demonstrated using plant proteins and the human interleukin-1 receptor-associated kinase family of proteins as example. The GCPred server is available at http://gcpred.com. Supplementary data are available at Bioinformatics online.

Accelerated Evolution of the Pituitary Adenylate Cyclase-Activating Polypeptide Precursor Gene During Human Origin

PubMed Central

Wang, Yin-qiu; Qian, Ya-ping; Yang, Su; Shi, Hong; Liao, Cheng-hong; Zheng, Hong-Kun; Wang, Jun; Lin, Alice A.; Cavalli-Sforza, L. Luca; Underhill, Peter A.; Chakraborty, Ranajit; Jin, Li; Su, Bing

2005-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. PMID:15834139

A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

PubMed Central

Chen, Mei-Yu; Long, Yu; Devreotes, Peter N.

1997-01-01

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA− null mutants, neither chemoattractant stimulation of intact cells nor GTPγS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPγS stimulation of adenylyl cyclase activity in piaA− lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac− piaA− double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. PMID:9389653

Structures of Human Golgi-resident Glutaminyl Cyclase and Its Complexes with Inhibitors Reveal a Large Loop Movement upon Inhibitor Binding*

PubMed Central

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H.-J.

2011-01-01

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05–1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC. PMID:21288892

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

PubMed

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

2011-04-08

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

NASA Astrophysics Data System (ADS)

Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

2017-01-01

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

PubMed Central

Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

2017-01-01

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids. PMID:28028245

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

PubMed

Svedova, Martina; Masin, Jiri; Fiser, Radovan; Cerny, Ondrej; Tomala, Jakub; Freudenberg, Marina; Tuckova, Ludmila; Kovar, Marek; Dadaglio, Gilles; Adkins, Irena; Sebo, Peter

2016-04-01

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.

PubMed

Graeff, R M; Walseth, T F; Fryxell, K; Branton, W D; Lee, H C

1994-12-02

Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior

PubMed Central

Bretscher, Andrew Jonathan; Kodama-Namba, Eiji; Busch, Karl Emanuel; Murphy, Robin Joseph; Soltesz, Zoltan; Laurent, Patrick; de Bono, Mario

2011-01-01

Summary Homeostatic control of body fluid CO2 is essential in animals but is poorly understood. C. elegans relies on diffusion for gas exchange and avoids environments with elevated CO2. We show that C. elegans temperature, O2, and salt-sensing neurons are also CO2 sensors mediating CO2 avoidance. AFD thermosensors respond to increasing CO2 by a fall and then rise in Ca2+ and show a Ca2+ spike when CO2 decreases. BAG O2 sensors and ASE salt sensors are both activated by CO2 and remain tonically active while high CO2 persists. CO2-evoked Ca2+ responses in AFD and BAG neurons require cGMP-gated ion channels. Atypical soluble guanylate cyclases mediating O2 responses also contribute to BAG CO2 responses. AFD and BAG neurons together stimulate turning when CO2 rises and inhibit turning when CO2 falls. Our results show that C. elegans senses CO2 using functionally diverse sensory neurons acting homeostatically to minimize exposure to elevated CO2. PMID:21435556

Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction.

PubMed

Misono, Kunio S; Ogawa, Haruo; Qiu, Yue; Ogata, Craig M

2005-06-01

The atrial natriuretic peptide (ANP) receptor is a single-span transmembrane receptor that is coupled to its intrinsic intracellular guanylate cyclase (GCase) catalytic activity. To investigate the mechanisms of hormone binding and signal transduction, we have expressed the extracellular hormone-binding domain of the ANP receptor (ANPR) and characterized its structure and function. The disulfide-bond structure, state of glycosylation, binding-site residues, chloride-dependence of ANP binding, dimerization, and binding stoichiometry have been determined. More recently, the crystal structures of both the apoANPR dimer and ANP-bound complex have been determined. The structural comparison between the two has shown that, upon ANP binding, two ANPR molecules in the dimer undergo an inter-molecular twist with little intra-molecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains with essentially no change in the inter-domain distance. This movement alters the relative orientation of the two domains equivalent to counter-clockwise rotation of each by 24 degrees . These results suggest that transmembrane signaling by the ANP receptor is mediated by a novel hormone-induced rotation mechanism.

Role of the decreased nitric oxide bioavailability in the vascular complications of diabetes mellitus.

PubMed

Masha, Andi; Dinatale, Stefano; Allasia, Stefano; Martina, Valentino

2011-09-01

This mini-review takes into consideration the physiology, synthesis and mechanisms of action of the nitric oxide (NO) and, subsequently, the causes and effects of the NO bioavailability impairment. In diabetes mellitus the reduced NO bioavailability is caused by the increased free radicals production, secondary to hyperglycemia. The reactive oxygen species oxidize the cofactors of the nitric oxide synthase, diminishing their active forms and consequently leading to a decreased NO production. Furthermore the decreased concentration of reduced glutathione results in a diminished production of nitrosoglutathione. These molecules are important intermediates of the NO pathway and physiologically activate the soluble guanylate cyclase. Their decrease in oxidative states of the cell, therefore, leads to a reduced cGMP production which represents the principal molecule that carries out NO's major effects. Finally we considered the eventual therapeutic strategies to improve NO bioavailability by acting on the causes of its decrease. Therefore the treatments proposed are based on the possibility to counteract the oxidation and, in this context, the physiopathological mechanisms strongly support the treatment with thiols.

[Antiplatelet properties of nitrogen monoxide].

PubMed

Adrie, C

1996-11-01

Nitric (correction of nitrous) oxide (NO) plays a fundamental part in the haemostatic equilibrium between the endothelium and platelets, an equilibrium of established clinical importance in cardiovascular disease. NO stimulates the enzyme guanylate cyclase which is responsible for synthesis of GMPc, the increase of which results in platelet inhibition. Synthesis of NO may have endogenous auto or paracrine origine from platelets or endothelial cells and participates in the local regulation of platelet function in association with other products of endothelial or platelet synthesis. Exogenous administration is common in therapeutics either in molecules which release NO (nitrate derivatives, sodium nitropruside, molsidomine, etc) or by NO gas administered by inhalation. The antiplatelet effect of NO has been clearly demonstrated in vitro, in vivo or ex vivo, in animals and humans, and probably explains, at least partially, the efficacy of nitrate derivatives in ischaemic coronary artery disease. Nevertheless, the platelet inhibition observed with intravenous NO releasing drugs is associated with potentially harmful systemic hypotension. Platelet inhibition by inhalation of NO could be an alternative means of avoiding this unwanted effect.

Leber congenital amaurosis: from darkness to spotlight.

PubMed

Kaplan, Josseline

2008-09-01

Almost 150 years ago, Theodor Leber described a severe form of vision loss at or near birth which was later given his name. During the century that followed this description, ophthalmologists dedicated efforts to give an accurate definition of the disease but patients were neglected because of the inability of physicians to provide them with treatment. In the 90s, at the time of the Golden Age of Linkage, the first LCA locus was mapped to a human chromosome and shortly after identified as the gene for guanylate cyclase. This discovery was the spark that made the disease emerge from the shadows as illustrated by the flood of LCA genes identified in the following ten-year period. During the same time period, the clinical variability of the disease was rediscovered and an unexpected physiopathological heterogeneity demonstrated. In the beginning of the third millennium, LCA came out definitively from the tunnel to shine under the bright spotlights with the RPE65 gene therapy trial that succeeded to restore vision in a dog model and opened the door to gene therapy trials in humans.

Novel Pharmacological Therapies for Management of Chronic Constipation

PubMed Central

Gonzalez-Martinez, Marina A.; Mendez-Navarro, Jorge

2014-01-01

Chronic constipation is a common health problem that significantly affects the quality of life of patients and impacts in terms of costs; current treatments based on fiber and laxatives cause dissatisfaction to doctors and patients in more than half of the cases. New drugs are now available or in very advanced stages of research, with different and innovative mechanisms of action as prucalopride, lubiprostone, and linaclotide. Prucalopride an enterokinetic, is a selective high-affinity 5-hydroxytryptamine (5-HT)4 receptor agonist of serotonin that increases the peristaltic reflex and the colonic contractions; lubiprostone, a type 2 chlorine channel activator, or linaclotide, a guanylate cyclase-C agonist of enterocytes, both prosecretory agents, stimulate the secretion of fluid within the intestinal lumen. In general, these promising drugs have proven efficacy and safety as a specific therapeutic option in patients with chronic constipation. Yet the solution might not be sufficient for everybody and still without the ideal drug that might be useful in all cases, the pharmacological revolution for colonic motility disorders has arrived. PMID:24172177

Structural impact analysis of missense SNPs present in the uroguanylin gene by long-term molecular dynamics simulations.

PubMed

Marcolino, Antonio C S; Porto, William F; Pires, Állan S; Franco, Octavio L; Alencar, Sérgio A

2016-12-07

The guanylate cyclase activator 2B, also known as uroguanylin, is part of the guanylin peptide family, which includes peptides such as guanylin and lymphoguanylin. The guanylin peptides could be related to sodium absorption inhibition and water secretion induction and their dysfunction may be related to various pathologies such as chronic renal failure, congestive heart failure and nephrotic syndrome. Besides, uroguanylin point mutations have been associated with essential hypertension. However, currently there are no studies on the impact of missense SNPs on uroguanylin structure. This study applied in silico SNP impact prediction tools to evaluate the impact of uroguanylin missense SNPs and to filter those considered as convergent deleterious, which were then further analyzed through long-term molecular dynamics simulations of 1μs of duration. The simulations suggested that all missense SNPs considered as convergent deleterious caused some kind of structural change to the uroguanylin peptide. Additionally, four of these SNPs were also shown to cause modifications in peptide flexibility, possibly resulting in functional changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inversion Monophyly in African Anopheline Malaria Vectors

PubMed Central

Garcia, B. A.; Caccone, A.; Mathiopoulos, K. D.; Powell, J. R.

1996-01-01

The African Anopheles gambiae complex of six sibling species has many polymorphic and fixed paracentric inversions detectable in polytene chromosomes. These have been used to infer phylogenetic relationships as classically done with Drosophila. Two species, A. gambiae and A. merus, were thought to be sister taxa based on a shared X inversion designated X(ag). Recent DNA data have conflicted with this phylogenetic inference as they have supported a sister taxa relationship of A. gambiae and A. arabiensis. A possible explanation is that the X(ag) is not monophyletic. Here we present data from a gene (soluble guanylate cyclase) within the X(ag) that strongly supports the monophyly of the X(ag). We conjecture that introgression may be occurring between the widely sympatric species A. gambiae and A. arabiensis and that the previous DNA phylogenies have been detecting the introgression. Evidently, introgression is not uniform across the genome, and species-specific regions, like the X-chromosome inversions, do not introgress probably due to selective elimination in hybrids and backcrosses. PMID:8807303

Prevention and treatment of the chronic thromboembolic pulmonary hypertension.

PubMed

Pesavento, Raffaele; Prandoni, Paolo

2018-04-01

Chronic thromboembolic pulmonary hypertension (CTEPH) is an uncommon and late complication of pulmonary embolism resulting from misguided remodelling of residual pulmonary thromboembolic material and small-vessel arteriopathy. CTEPH is the only form of pulmonary hypertension (PH) potentially curable by pulmonary endarterectomy (PEA). Unfortunately, several patients have either an unacceptable risk-benefit ratio for undergoing the surgical intervention or develop persistent PH after PEA. Novel medical and endovascular therapies can be considered for them. The soluble guanylate cyclase stimulator riociguat is recommended for the treatment of patients with inoperable disease or with recurrent/persistent PH after PEA. Other drugs developed for the treatment of other forms of PH, as prostanoids, phosphodiesterase-5 inhibitors and endothelin receptor antagonists have been used in the treatment of CTEPH, with limited benefit. Balloon pulmonary angioplasty is a novel and promising technique and is progressively emerging from the pioneering phase. Highly specialized training level and complex protocols of postoperative care are mandatory to consolidate the technical success of the surgical and endovascular intervention. Copyright © 2018 Elsevier Ltd. All rights reserved.

SYMPOSIUM REPORT: An Evidence-Based Approach to IBS and CIC: Applying New Advances to Daily Practice: A Review of an Adjunct Clinical Symposium of the American College of Gastroenterology Meeting October 16, 2016 • Las Vegas, Nevada.

PubMed

Chey, William D

2017-02-01

Many nonpharmacologic and pharmacologic therapies are available to manage irritable bowel syndrome (IBS) and chronic idiopathic constipation (CIC). The American College of Gastroenterology (ACG) regularly publishes reviews on IBS and CIC therapies. The most recent of these reviews was published by the ACG Task Force on the Management of Functional Bowel Disorders in 2014. The key objective of this review was to evaluate the efficacy of therapies for IBS or CIC compared with placebo or no treatment in randomized controlled trials. Evidence-based approaches to managing diarrhea-predominant IBS include dietary measures, such as a diet low in gluten and fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs); loperamide; antispasmodics; peppermint oil; probiotics; tricyclic antidepressants; alosetron; eluxadoline, and rifaximin. Evidence-based approaches to managing constipation-predominant IBS and CIC include fiber, stimulant laxatives, polyethylene glycol, selective serotonin reuptake inhibitors, lubiprostone, and guanylate cyclase agonists. With the growing evidence base for IBS and CIC therapies, it has become increasingly important for clinicians to assess the quality of evidence and understand how to apply it to the care of individual patients.

SYMPOSIUM REPORT: An Evidence-Based Approach to IBS and CIC: Applying New Advances to Daily Practice

PubMed Central

Chey, William D.

2017-01-01

Many nonpharmacologic and pharmacologic therapies are available to manage irritable bowel syndrome (IBS) and chronic idiopathic constipation (CIC). The American College of Gastroenterology (ACG) regularly publishes reviews on IBS and CIC therapies. The most recent of these reviews was published by the ACG Task Force on the Management of Functional Bowel Disorders in 2014. The key objective of this review was to evaluate the efficacy of therapies for IBS or CIC compared with placebo or no treatment in randomized controlled trials. Evidence-based approaches to managing diarrhea-predominant IBS include dietary measures, such as a diet low in gluten and fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs); loperamide; antispasmodics; peppermint oil; probiotics; tricyclic antidepressants; alosetron; eluxadoline, and rifaximin. Evidence-based approaches to managing constipation-predominant IBS and CIC include fiber, stimulant laxatives, polyethylene glycol, selective serotonin reuptake inhibitors, lubiprostone, and guanylate cyclase agonists. With the growing evidence base for IBS and CIC therapies, it has become increasingly important for clinicians to assess the quality of evidence and understand how to apply it to the care of individual patients. PMID:28729815

Guanylyl cyclase-dependent chemotaxis of endothelial cells in response to nitric oxide gradients.

PubMed

Isenberg, Jeff S; Ridnour, Lisa A; Thomas, Douglas D; Wink, David A; Roberts, David D; Espey, Michael Graham

2006-03-15

Nitric oxide (NO) is an important regulator of angiogenesis and neovascularization. The nature of endothelial cell motility responses to NO was examined using a Boyden chamber method. NO generated via decomposition of either DEA/NO or DETA/NO produced increases in human umbilical vein endothelial cell (HUVEC) chemotaxis, which were completely abrogated by ODQ, a soluble guanylyl cyclase inhibitor. Measurements of NO either directly by chemiluminescence or its chemistry with diaminofluorescein revealed that chemotaxis was driven by subtle NO gradients between the lower and the upper wells in this system. In addition to diffusion and volatilization from the upper chambers, the data showed that HUVEC consumption of NO contributed to these sustained gradients. Comparison of DEA/NO- and DETA/NO-mediated responses suggested that the persistence of spatial NO gradients is as significant as the absolute magnitude of NO exposure per unit time. The findings suggest that subnanomolar NO gradients are sufficient to mobilize endothelial cell migration into hypoxic tissue during neovascularization events, such as in wound healing and cancer.

Molecular Characterization of Tick Salivary Gland Glutaminyl Cyclase

PubMed Central

Adamson, Steven W.; Browning, Rebecca E.; Chao, Chien-Chung; Bateman, Robert C.; Ching, Wei-Mei; Karim, Shahid

2013-01-01

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 blood-meal of adult female ticks prior to fast feeding phases in both I. scapularis and A. maculatum suggesting a functional link with blood meal 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

The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria.

PubMed

Sugimoto, Hiroki; Kusumi, Kensuke; Noguchi, Ko; Yano, Masahiro; Yoshimura, Atsushi; Iba, Koh

2007-11-01

Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.

Acute blood pressure effects of YC-1-induced activation of soluble guanylyl cyclase in normotensive and hypertensive rats

PubMed Central

Rothermund, Lars; Friebe, Andreas; Paul, Martin; Koesling, Doris; Kreutz, Reinhold

2000-01-01

We used YC-1 as a pharmacological tool to investigate the short-term blood pressure effects of NO-independent activation of sGC in normotensive and hypertensive rats. Four groups of normotensive Wistar-Kyoto rats were treated by i.v. injection with vehicle (V), YC-1 (YC-1), sodium nitroprusside (SNP), or YC-1 and SNP (YC-1+SNP). Hypertension was induced in four additional groups of WKY rats by 3 weeks of oral treatment with L-NAME. These animals were investigated with the same protocol as the normotensive animals: L-NAME/V, L-NAME/YC-1, L-NAME/SNP, L-NAME/YC-1+SNP. YC-1 lowered mean arterial blood pressure (MAP) in normotensive and hypertensive animals similarly to SNP alone (P<0.05, respectively). The combination of YC-1 with SNP caused a strong decrease of MAP in both the hypertensive and normotensive animals (P<0.05, respectively). SNP with YC-1 also induced a pronounced cyclic GMP increase in the aorta. This study shows for the first time the blood pressure lowering potential of bimodal targeting of the NO-sGC-system. PMID:10807655

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

PubMed Central

Shih, Yan-Ping; Chou, Chi-Chi; Chen, Yi-Ling; Huang, Kai-Fa; Wang, Andrew H.- J.

2014-01-01

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities. PMID:24733552

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

PubMed Central

Bagley, Elena E.

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

PubMed

Bagley, Elena E

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

PubMed Central

Chia, Karin K. M.; Liu, Chia-Chi; Hamilton, Elisha J.; Garcia, Alvaro; Fry, Natasha A.; Hannam, William; Figtree, Gemma A.

2015-01-01

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22phox and p47phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. PMID:26084308

The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

PubMed Central

Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

2016-01-01

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

Antidepressant-like effects induced by NMDA receptor blockade and NO synthesis inhibition in the ventral medial prefrontal cortex of rats exposed to the forced swim test.

PubMed

Pereira, Vitor Silva; Romano, Angélica; Wegener, Gregers; Joca, Sâmia R L

2015-07-01

Systemic treatment with NMDA receptor (NMDAR) antagonists, inhibitors of neuronal nitric oxide synthase (nNOS) or of soluble guanylyl cyclase (sGC), induce antidepressant-like effects in rats. Increased levels of glutamate and nitric oxide (NO) in the medial prefrontal cortex (MPFC) of stressed animals have been described in the literature. However, the role of the NMDAR-nNOS-sGC pathway of the MPFC in the mediation of forced swim-induced behaviors remains unclear. The aim of this work was to test the hypothesis that the inhibition of the NMDAR-nNOS-sGC pathway in the ventral MPFC (infralimbic (IL) or prelimbic (PL)) would elicit antidepressant-like effects in the forced swim test (FST). Rats implanted with cannulae aimed at the PL or the IL were exposed to the FST and injected with LY235959 (NMDAR antagonist), NPA (nNOS inhibitor), ODQ (sGC inhibitor), or carboxy-PTIO (NO scavenger). Additional groups received the AMPA antagonist, NBQX, before the effective doses of LY235959 or NPA. LY235959 administration into PL or IL before the FS pretest produced no effects. Administration of LY235959 (3 and 10 nmol/0.2 μL) after pretest was effective only when administered into the PL. However, the administration of NPA (0.01 nmol/0.2 μL), c-PTIO (1.0 nmol/0.2 μL), and ODQ (1.0 nmol/0.2 μL) into the PL or IL before the FST produced antidepressant-like effects. NBQX blocked the antidepressant-like effect of LY235959 but not of NPA. Blocking NMDAR or NO signaling in the vMPFC, either in the IL or the PL, induces antidepressant-like effects in the rat FST. These effects seemingly occur through independent mechanisms, since NBQX blocked the former effect but not the latter.

The inhibitory effect of 3-amino-1,2,4-triazole on relaxation induced by hydroxylamine and sodium azide but not hydrogen peroxide or glyceryl trinitrate in rat aorta.

PubMed Central

Mian, K. B.; Martin, W.

1995-01-01

1. In this study we investigated the role of catalase in relaxation induced by hydroxylamine, sodium azide, glyceryl trinitrate and hydrogen peroxide in isolated rings of rat aorta. 2. Hydrogen peroxide (1 microM-1 mM)-induced concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings. In endothelium-denuded rings, however, higher concentrations (30 microM-1 mM) of hydrogen peroxide were required to produce relaxation. The endothelium-dependent component of hydrogen peroxide-induced relaxation was abolished following pretreatment with N(O)-nitro-L-arginine methyl ester (L-NAME, 30 microM). L-NAME (30 microM) had no effect, however, on hydrogen peroxide-induced relaxation in endothelium-denuded rings. 3. Pretreatment of endothelium-denuded rings with catalase (1000 u ml-1) blocked relaxation induced by hydrogen peroxide (10 microM-1 mM). The ability of catalase to inhibit hydrogen peroxide-induced relaxation was partially blocked following incubation with 3-amino-1,2, 4-triazole (AT, 50 mM) for 30 min and completely blocked at 90 min. 4. Pretreatment of endothelium-denuded rings with methylene blue (MeB, 30 microM) inhibited relaxation induced by hydrogen peroxide (10 microM-1 mM), sodium azide (1-300 nM), hydroxylamine (1-300 nM) and glyceryl trinitrate (1-100 nM) suggesting that each acted by stimulation of soluble guanylate cyclase. 5. Pretreatment of endothelium-denuded rings with AT (1-50 mM, 90 min) to inhibit endogenous catalase blocked relaxation induced by sodium azide (1-300 nM) and hydroxylamine (1-300 nM) but had no effect on relaxation induced by hydrogen peroxide (10 microM-1 mM) or glyceryl trinitrate (1-100 nM). 6. In a cell-free system, incubation of sodium azide (10 microM-3 mM) and hydroxylamine (10 microM-30 mM) but not glyceryl trinitrate (10 microM-1 mM) with catalase (1000 u ml-1) in the presence of hydrogen peroxide (1 mM) led to production of nitrite, a major breakdown product of nitric oxide. AT (1

Characterisation of Two Oxidosqualene Cyclases Responsible for Triterpenoid Biosynthesis in Ilex asprella

PubMed Central

Zheng, Xiasheng; Luo, Xiuxiu; Ye, Guobing; Chen, Ye; Ji, Xiaoyu; Wen, Lingling; Xu, Yaping; Xu, Hui; Zhan, Ruoting; Chen, Weiwen

2015-01-01

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship. PMID:25664861

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

PubMed Central

Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

2014-01-01

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

Activity of adenylyl cyclase and protein kinase A contributes to morphine-induced spinal apoptosis.

PubMed

Lim, Grewo; Wang, Shuxing; Lim, Jeong-Ae; Mao, Jianren

2005-12-02

Our previous study has shown that chronic morphine exposure induces neuronal apoptosis within the spinal cord dorsal horn; however, the mechanisms of morphine-induced apoptosis remain unclear. Here we examined whether adenylyl cyclase (AC) and protein kinase A (PKA) would play a role in this process. Intrathecal morphine regimen (10 microg, twice daily x 7 days) that resulted in antinociceptive tolerance induced spinal apoptosis as revealed by in situ terminal deoxynucleotidyl transferase (TdT)-UTP-biotin nick end labeling (TUNEL). The TUNEL-positive cells were detected primarily in the superficial laminae of the spinal cord dorsal horn, which was associated with an increase in the expression of activated caspase-3 and mitogen-activated protein kinase (MAPK) within the same spinal region. Co-administration of morphine with a broad AC inhibitor (ddA), a PKA inhibitor (H89), or a MAPK inhibitor (PD98059) substantially reduced the number of TUNEL-positive cells, as compared with the morphine alone group. The results indicate that the spinal AC and PKA pathway through intracellular MAPK may be contributory to the cellular mechanisms of morphine-induced apoptosis.

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

PubMed

Marondedze, Claudius; Wong, Aloysius; Thomas, Ludivine; Irving, Helen; Gehring, Chris

2017-01-01

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

Use of Single-Layer g-C3N4/Ag Hybrids for Surface-Enhanced Raman Scattering (SERS)

PubMed Central

Jiang, Jizhou; Zou, Jing; Wee, Andrew Thye Shen; Zhang, Wenjing

2016-01-01

Surface-enhanced Raman scattering (SERS) substrates with high activity and stability are desirable for SERS sensing. Here, we report a new single atomic layer graphitic-C3N4 (S-g-C3N4) and Ag nanoparticles (NPs) hybrid as high-performance SERS substrates. The SERS mechanism of the highly stable S-g-C3N4/Ag substrates was systematically investigated by a combination of experiments and theoretical calculations. From the results of XPS and Raman spectroscopies, it was found that there was a strong interaction between S-g-C3N4 and Ag NPs, which facilitates the uniform distribution of Ag NPs over the edges and surfaces of S-g-C3N4 nanosheets, and induces a charge transfer from S-g-C3N4 to the oxidizing agent through the silver surface, ultimately protecting Ag NPs from oxidation. Based on the theoretical calculations, we found that the net surface charge of the Ag atoms on the S-g-C3N4/Ag substrates was positive and the Ag NPs presented high dispersibility, suggesting that the Ag atoms on the S-g-C3N4/Ag substrates were not likely to be oxidized, thereby ensuring the high stability of the S-g-C3N4/Ag substrate. An understanding of the stability mechanism in this system can be helpful for developing other effective SERS substrates with long-term stability. PMID:27687573

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

PubMed

Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

2012-06-01

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adenovirus-mediated IL-24 expression enhances the chemosensitivity of multidrug-resistantgastric cancer cells to cisplatin.

PubMed

Mao, Zonglei; Bian, Guochun; Sheng, Weihua; He, Songbin; Yang, Jicheng; Dong, Xiaoqiang

2013-11-01

Chemotherapy is one of the commonly used strategies in gastric cancer, especially for unresectable patients, but it becomes insensitive to repeated administration of even the most effective chemotherapeutic agents, such as cisplatin. Given this, there is an urgent need for developing chemosensitizers to overcome acquired resistance to chemotherapeutic agents. Interleukin-24 (IL-24), a cytokine-tumor suppressor, shows broad-spectrum and tumor-specific antitumor properties, and studies have demonstrated that IL-24 could conspicuously restore the chemosensitivity of MDR cancer cells. Herein, we developed a human MDR gastric cancer cell subline, SGC7901/CDDP, by repeated selection of resistant clones of parental sensitive cells, and further investigated the chemosensitizing effects and the underlying mechanisms of adenovirus-mediated IL-24 (Ad-IL-24) gene therapy plus CDDP for the human MDR gastric cancer cells SGC7901/CDDP in vitro and in vivo. The results demonstrated that the expression of IL-24 mRNA and protein was profoundly downregulated in SGC7901/CDDP cells by RT-PCR and western blot analysis. In addition, the cell viability assay showed that the IC50 of SGC7901/CDDP cells to CDDP, 5-FU, ADM and MTX was significantly enhanced compared to parental sensitive SGC7901 cells. Ad-IL-24-induced IL-24 overexpression decreased the IC50 of the above agents (not MTX), induced G2/M cell cycle arrest, and Ad-IL-24 plus CDDP elicited significant apoptosis and tumor suppression of SGC7901/CDDP cells in vitro and SGC7901/CDDP cell xenograft tumors in vivo, respectively. Moreover, our results demonstrated that the mechanisms of Ad-IL-24-elicited chemosensitizing effects were closely associated with a substantial upregulation of Bax and downregulation of P-gp and Bcl-2 in SGC7901/CDDP cells in vitro and SGC7901/CDDP xenograft tissues in vivo. Thus, this study indicates that overexpression of IL-24 gene can significantly promote chemosensitivity in MDR phenotype SGC7901

Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin.

PubMed

Springer, Tzvia I; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L

2014-10-10

Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM. Copyright © 2014 Elsevier Inc. All rights reserved.

Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

PubMed

Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

2013-03-06

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

NASA Astrophysics Data System (ADS)

Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

2010-10-01

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

Cavity electromagnetically induced transparency via spontaneously generated coherence

NASA Astrophysics Data System (ADS)

Tariq, Muhammad; Ziauddin, Bano, Tahira; Ahmad, Iftikhar; Lee, Ray-Kuang

2017-09-01

A four-level N-type atomic ensemble enclosed in a cavity is revisited to investigate the influence of spontaneous generated coherence (SGC) on transmission features of weak probe light field. A weak probe field is propagating through the cavity where each atom inside the cavity follows four-level N-type atom-field configuration of rubidium (?) atom. We use input-output theory and study the interaction of atomic ensemble and three cavity fields which are coupled to the same cavity mode. A SGC affects the transmission properties of weak probe light field due to which a transparency window (cavity EIT) appears. At resonance condition the transparency window increases with increasing the SGC in the system. We also studied the influence of the SGC on group delay and investigated magnitude enhancement of group delay for the maximum SGC in the system.

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes

PubMed Central

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex

2016-01-01

ABSTRACT DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. IMPORTANCE Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes.

PubMed

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex; Becker, Anke

2016-09-15

DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c-di-GMP-producing diguanylate

A Comparative Analysis of the Sugar Phosphate Cyclase Superfamily Involved in Primary and Secondary Metabolism

PubMed Central

Wu, Xiumei; Flatt, Patricia M.; Schlörke, Oliver; Zeeck, Axel; Dairi, Tohru

2011-01-01

Sugar Phosphate Cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, e.g., aromatic amino acids, and clinically relevant secondary metabolites, e.g., aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically-labeled cyclitols revealed that cetoniacytone A, a unique C7N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. Using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homolog gene, cetA, was isolated from the cetoniacytone producer. CetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that may regulate the biosynthesis of a novel set of secondary metabolites. PMID:17195255

Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs.

PubMed

Pinto, Cibele; Papa, Dan; Hübner, Melanie; Mou, Tung-Chung; Lushington, Gerald H; Seifert, Roland

2008-04-01

Adenylyl cyclase (AC) isoforms 1 to 9 are differentially expressed in tissues and constitute an interesting drug target. ACs 1 to 8 are activated by the diterpene, forskolin (FS). It is unfortunate that there is a paucity of AC isoform-selective activators. To develop such compounds, an understanding of the structure/activity relationships of diterpenes is necessary. Therefore, we examined the effects of FS and nine FS analogs on ACs 1, 2, and 5 expressed in Spodoptera frugiperda insect cells. Diterpenes showed the highest potencies at AC1 and the lowest potencies at AC2. We identified full agonists, partial agonists, antagonists, and inverse agonists, i.e., diterpenes that reduced basal AC activity. Each AC isoform exhibited a distinct pharmacological profile. AC2 showed the highest basal activity of all AC isoforms and highest sensitivity to inverse agonistic effects of 1-deoxy-forskolin, 7-deacetyl-1,9-dideoxy-forskolin, and, particularly, BODIPY-forskolin. In contrast, BODIPY-forskolin acted as partial agonist at the other ACs. 1-Deoxy-forskolin analogs were devoid of agonistic activity at ACs but antagonized the effects of FS in a mixed competitive/noncompetitive manner. At purified catalytic AC subunits, BODIPY-forskolin acted as weak partial agonist/strong partial antagonist. Molecular modeling revealed that the BODIPY group rotates promiscuously outside of the FS-binding site. Collectively, ACs are not uniformly activated and inhibited by FS and FS analogs, demonstrating the feasibility to design isoform-selective FS analogs. The two- and multiple-state models, originally developed to conceptualize ligand effects at G-protein-coupled receptors, can be applied to ACs to explain certain experimental data.

Cyclase inhibitor tripropylamine significantly enhanced lycopene accumulation in Blakeslea trispora.

PubMed

Wang, Yanlong; Chen, Xiwen; Hong, Xiao; Du, Shipeng; Liu, Chunxiao; Gong, Wenfang; Chen, Defu

2016-11-01

Lycopene is a member of carotenoids that exhibits strong antioxidant activity. In this study, on the basis of screening suitable strain combination [ATCC 14271(+) and ATCC 14272(-)] and establishing the optimal inoculation proportion of mated culture (1/2, +/-, w/w) for carotenoid production, the efficiency of compounds, mainly tertiary amines, on enhancing the lycopene content of Blakeslea trispora was systematically assessed. Of these compounds, tripropylamine showed the best enhancing effect, and then sequentially followed by triethylamine, tributylamine, trimethylamine, diisopropylamine, and isopropylamine. After treated with 1.8 g/L tripropylamine for two days, the lycopene proportion was increased from 1.7% to 90.1%, while the β-carotene proportion was decreased from 91.1% to 6.4% of the total carotenoids. In this case, the lycopene and total carotenoid contents were increased to 83.2 and 92.4 mg/gDW, which were 315.8- and 5.9-fold of that of the untreated control, respectively; while the growth of mycelia was only decreased at 6.0 g/L tripropylamine. Gene expression analysis showed that all the tested genes, especially genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and isopentenyl pyrophosphate isomerase (ipi) in mevalonate pathway, as well as phytoene desaturase (carB) in carotenoid biosynthesis process were upregulated. Therefore, tripropylamine enhanced lycopene content of B. trispora by inhibiting the cyclase activity, and by upregulating the expression of genes associated with terpenoid biosynthesis. Besides, a possible association between the structure and the lycopene-enhancing capability of these compounds was also discussed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

PubMed Central

Tlili, Mounira; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Wurtz, Olivier

2015-01-01

The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

PubMed

Celano, M; Arturi, F; Presta, I; Bruno, R; Scarpelli, D; Calvagno, M G; Cristofaro, C; Bulotta, S; Giannasio, P; Sacco, R; Filetti, S; Russo, D

2003-05-30

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

Natriuretic Peptide Receptor Guanylyl Cyclase-A Protects Podocytes from Aldosterone-Induced Glomerular Injury

PubMed Central

Ogawa, Yoshihisa; Yokoi, Hideki; Kasahara, Masato; Mori, Kiyoshi; Kato, Yukiko; Kuwabara, Takashige; Imamaki, Hirotaka; Kawanishi, Tomoko; Koga, Kenichi; Ishii, Akira; Tokudome, Takeshi; Kishimoto, Ichiro; Sugawara, Akira; Nakao, Kazuwa

2012-01-01

Natriuretic peptides produced by the heart in response to cardiac overload exert cardioprotective and renoprotective effects by eliciting natriuresis, reducing BP, and inhibiting cell proliferation and fibrosis. These peptides also antagonize the renin-angiotensin-aldosterone system, but whether this mechanism contributes to their renoprotective effect is unknown. Here, we examined the kidneys of mice lacking the guanylyl cyclase-A (GC-A) receptor for natriuretic peptides under conditions of high aldosterone and high dietary salt. After 4 weeks of administering aldosterone and a high-salt diet, GC-A knockout mice, but not wild-type mice, exhibited accelerated hypertension with massive proteinuria. Aldosterone-infused GC-A knockout mice had marked mesangial expansion, segmental sclerosis, severe podocyte injury, and increased oxidative stress. Reducing the BP with hydralazine failed to lessen such changes; in contrast, blockade of the renin-angiotensin-aldosterone system markedly reduced albuminuria, ameliorated podocyte injury, and reduced oxidative stress. Furthermore, treatment with the antioxidant tempol significantly reduced albuminuria and abrogated the histologic changes. In cultured podocytes, natriuretic peptides inhibited aldosterone-induced mitogen-activated protein kinase phosphorylation. Taken together, these results suggest that renoprotective properties of the endogenous natriuretic peptide/GC-A system may result from the local inhibition of the renin-angiotensin-aldosterone system and oxidative stress in podocytes. PMID:22652704