scholarly journals Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum

1995 ◽  
Vol 306 (3) ◽  
pp. 801-809 ◽  
Author(s):  
Y Shakur ◽  
M Wilson ◽  
L Pooley ◽  
M Lobban ◽  
S L Griffiths ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Laser Scanning ◽  
Plasma Membranes ◽  
High Sensitivity ◽  
Fold Increase ◽  
Camp Phosphodiesterase ◽  
Cos Cells ◽  
Denaturation Kinetics ◽  
Ionic Detergent ◽  
Immunofluorescence Studies

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat ‘dunc-like’ cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5′-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum

1986 ◽  
Vol 235 (2) ◽  
pp. 491-498 ◽  
Author(s):  
P Thiyagarajah ◽  
S C Lim
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclic Amp ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Parotid Glands ◽  
Tissue Homogenates ◽  
Rat Parotid

A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [(Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.

scholarly journals Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

1990 ◽  
Vol 265 (10) ◽  
pp. 5840-5846
Author(s):  
M M Van Lookeren Campagne ◽  
E Wu ◽  
R D Fleischmann ◽  
M M Gottesman ◽  
K W Chason ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Mammalian Cells ◽  
Camp Phosphodiesterase

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

scholarly journals Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

1982 ◽  
Vol 202 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Clive J. Dix ◽  
Matthias Schumacher ◽  
Brian A. Cooke
Keyword(s):  
Cyclic Amp ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Linear Increase ◽  
Guanine Nucleotide ◽  
Production Rates ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Guanine Nucleotide Binding ◽  
Guanine Nucleotide Regulatory Protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

scholarly journals Cyclic AMP compartmentation due to increased cAMP‐phosphodiesterase activity in transgenic mice with a cardiac‐directed expression of the human adenylyl cyclase type 8 (AC8)

2003 ◽  
Vol 17 (11) ◽  
pp. 1380-1391 ◽  
Author(s):  
Marie Georget ◽  
Philippe Mateo ◽  
Grégoire Vandecasteele ◽  
Larissa Lipskaia ◽  
Nicole Defer ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Transgenic Mice ◽  
Adenylyl Cyclase ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase

scholarly journals Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors

1997 ◽  
Vol 326 (2) ◽  
pp. 545-551 ◽  
Author(s):  
Edward S. TOBIAS ◽  
Enrique ROZENGURT ◽  
John M. C. CONNELL ◽  
Miles D. HOUSLAY
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Kinase D ◽  
Intracellular Camp ◽  
Camp Production ◽  
Camp Phosphodiesterase ◽  
Glucagon Receptor ◽  
Cos Cells ◽  
Protein Receptor ◽  
Dose Dependent Fashion

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8±0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38–42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-α, PKC-βII and PKC-ϵ) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

Simplex Optimized Acetylacetone Method for Formaldehyde

1973 ◽  
Vol 56 (6) ◽  
pp. 1496-1502
Author(s):  
Fred P Czech
Keyword(s):  
Standard Deviation ◽  
Experimental Design ◽  
Design Process ◽  
Analytical Methods ◽  
High Sensitivity ◽  
Fold Increase ◽  
Chromotropic Acid ◽  
High Quality ◽  
Relative Standard ◽  
Aoac Method

Abstract The AOAC colorimetric acetylacetone method for formaldehyde in sugar products is optimized by means of the simplex experimental design process. The resultant method is almost 4 times as sensitive as the original AOAC method. It is about 33% more sensitive than the simplex optimized J-acid procedure and 45% more sensitive than the simplex optimized chromotropic acid method and, thus, represents one of the most sensitive methods now available. The average relative standard deviation is about ±2.7%. The limit of detectability is estimated to be 30 ppb. The didactic exposition of the simplex optimization process reported earlier is applied to the AOAC acetyl-acetone method. Further insights into simplex operations are provided and certain advantages of simplex application are pointed out. It is shown that, by sacrificing the high sensitivity of the optimized method the parameters of the new method result in about a 10-fold increase in analytical speed. Further application of kinetic considerations and the advantages of high quality, productivity, and economy in chemical analytical methods by means of the simplex experimental design process are described.

Immunogenicity of Golimumab and its Clinical Relevance in Patients With Ulcerative Colitis

2019 ◽  
Vol 25 (9) ◽  
pp. 1532-1540 ◽  
Author(s):  
Omoniyi J Adedokun ◽  
George R Gunn ◽  
Jocelyn H Leu ◽  
Cynthia Gargano ◽  
Zhenhua Xu ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
False Negative ◽  
Injection Site Reaction ◽  
High Sensitivity ◽  
Fold Increase ◽  
Drug Interference ◽  
Negative Results ◽  
Drug Concentrations ◽  
Antidrug Antibody ◽  
Antibody Titers

Abstract Background Antidrug antibody (ADA) detection with standard bridging enzyme immunoassays (EIA) can yield false-negative results or underestimate titers through drug interference. A more sensitive assay was needed to determine clinical impact of antigolimumab antibodies. Methods A high-sensitivity, drug-tolerant EIA (DT-EIA) was developed and cross-validated against the original EIA, and samples from induction/maintenance studies in golimumab-treated patients with ulcerative colitis were analyzed for ADAs using both methods. Immunogenicity results were compared, and pharmacokinetic, efficacy, and safety associations were evaluated. Results An 8-fold increase in ADA-positive patients (21.8% DT-EIA vs 2.8% EIA) reflected DT-EIA improved sensitivity and drug tolerance. Most newly detected ADA-positive patients (using DT-EIA) had low antibody titers, whereas most with high antibody titers were ADA-positive with original EIA. With DT-EIA, week 44 median trough serum golimumab concentrations among ADA-positive patients were approximately half vs ADA-negative (0.51 vs 0.85 µg/mL [50 mg q4w]; 0.85 vs 1.60 µg/mL [100 mg q4w]). Antidrug antibody impact on golimumab concentrations was more notable at titers ≥1:100. During induction, ADAs had no notable impact on efficacy. During maintenance, proportions of patients maintaining clinical response through week 54 were lower using DT-EIA: 38.1% ADA-positive and 52.8% ADA-negative. Antidrug antibody status had no impact on injection-site reaction incidence. Conclusions A more sensitive DT-EIA identified higher proportions of ADA-positive patients. A trend of decreasing drug concentrations with increasing ADA titers was observed. Pharmacokinetic impact was better elucidated with DT-EIA. Although development of ADA did not preclude efficacy, a trend toward decreased efficacy in ADA-positive vs ADA-negative patients was observed during maintenance treatment. Antidrug antibody status did not impact safety.

scholarly journals Laser-metal interaction dynamics during additive manufacturing resolved by detection of thermally-induced electron emission

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Philip J. DePond ◽  
John C. Fuller ◽  
Saad A. Khairallah ◽  
Justin R. Angus ◽  
Gabe Guss ◽  
...  
Keyword(s):  
Additive Manufacturing ◽  
Laser Scanning ◽  
Surface Defects ◽  
Thermal Emission ◽  
Thermionic Emission ◽  
High Sensitivity ◽  
Precise Determination ◽  
Diagnostic Methods ◽  
In Situ Monitoring ◽  
Interaction Dynamics

AbstractIn situ monitoring is required to improve the understanding and increase the reliability of additive manufacturing methods such as laser powder bed fusion (LPBF). Current diagnostic methods for LPBF capture optical images, X-ray radiographs, or measure the emission of thermal or acoustic signals from the component. Herein, a methodology based on the thermal emission of electrons - thermionic emission - from the metal surface during LPBF is proposed which can resolve laser-material interaction dynamics. The high sensitivity of thermionic emission to surface temperature and surface morphology is revealed to enable precise determination of the transition between conduction and keyhole mode melting regimes. Increases in thermionic emission are correlated to laser scanning conditions that give rise to pore formation and regions where surface defects are pronounced. The information presented here is a critical step in furthering our understanding and validation of laser-based metal additive manufacturing.

Dynamics of nucleotides in distal nephron of mice with nephrogenic diabetes insipidus

1986 ◽  
Vol 250 (1) ◽  
pp. F151-F158 ◽  
Author(s):  
E. Kusano ◽  
A. N. Yusufi ◽  
N. Murayama ◽  
J. Braun-Werness ◽  
T. P. Dousa
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Camp Phosphodiesterase ◽  
Camp Content ◽  
Medullary Thick Ascending Limb ◽  
Atp Levels ◽  
Collecting Tubules ◽  
Camp Levels

In mice with hereditary nephrogenic diabetes insipidus (NDI), the high activity of cAMP-phosphodiesterase (cAMP-PDIE) in medullary collecting tubules (MCT) prevents the increase in cAMP content in response to vasopressin [Arg8]vasopressin (AVP). Even when the cAMP response to AVP is partly corrected by cAMP-PDIE inhibitor 1-methyl-3-isobutylxanthine (MIX), under all tested conditions the cAMP levels in MCT of NDI mice remained much lower than in controls (B. A. Jackson, R. M. Edwards, H. Valtin, and T. P. Dousa, J. Clin. Invest. 66: 110-122, 1980). In the present study, we explored which factors may account for this defect. We determined contents of ATP, nicotinamide adenine dinucleotide (NAD), and the levels of cAMP in MCT and in medullary thick ascending limb of Henle's loop (MAL) microdissected from control and NDI mice. In the presence of 1 microM AVP and 0.05 mM MIX, the cAMP levels accumulated in MCT of NDI mice were four times lower compared with controls, but the levels of ATP and NAD were not different. ATP levels in MAL of NDI mice were slightly (delta -23%) lower than in MAL from controls, and in distal convoluted tubules (DCT) of NDI mice the ATP levels were also decreased (delta -49%). Although AVP alone had little effect on cAMP levels in mouse MAL in the presence of 0.1 mM forskolin, the AVP elicited a 20-fold increase of cAMP of both the control and NDI mice. Addition of 0.1 mM forskolin further increased the cAMP accumulation in MCT incubated with AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

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