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Sample records for adenosine deaminase activity

  1. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273565

  2. Serum adenosine deaminase and its isoenzyme activities in pregnancy

    Göksel Bahadır

    2011-12-01

    Full Text Available Objective: ADA is widely distributed in human tissues, which may contribute to the maturation of the immunological system, especially the proliferation and differentiation of lymphoid cells, and seems to be critical at different stages of the maturation process. The activity of ADA changes in diseases characterized by the alteration of cell-mediated immunity. In this study we examined changes in serum total ADA activity and the patterns of two ADA isoenzymes, ADA-1 and ADA-2, in healthy pregnant women, and evaluated the possible role of the alteration of cell-mediated immunity during pregnancy as causes of changes in ADA activity. Materials and Methods: We measured serum activities of total ADA, ADA-1 and ADA-2 in healthy pregnant women (n=129 and age-matched healthy nonpregnant women (n=42. We divided the study group into three different subgroups: first trimester, second trimester and third trimester. Results: Serum ADA, ADA-1 and ADA-2 activities in healthy pregnant women were significantly lower than in nonpregnant women (p<0.001, p<0.001 and p<0.01 respectively. ADA (p<0.001 and ADA-2 (p<0.001 activities in the first trimester were significantly lower than in the control group. However, there were no significant differences between the first trimester and control group according to their ADA-1 activities (p=0.016. ADA (p<0.001, ADA-1 (p<0.001 and ADA-2 (p<0.008 activities in the second trimester were significantly lower than in the control group. Combined trisomy 21 risk, biochemical trisomy 21 risk, age risk and trisomy 18 + Nuchal translucency (NT risk were calculated using a first trimester screening test in 63 pregnant women. Furthermore, trisomy 21 risk, age risk and trisomy 18 risk were calculated by triple test in 52 pregnant women. ADA, ADA-1 and ADA-2 activities were not significantly correlated with risks in the first trimester screening test. ADA-1 activity was slightly significantly negative correlated with age risk (r= -0

  3. Serum adenosine deaminase activity and its isoenzyme in patients treated for tuberculosis

    Objective: Increased serum adenosine deaminase (ADA) activity, mainly associated with tuberculosis can also occur in a number of other diseases thus negatively affecting the diagnostic utility of ADA measurements in tuberculosis. The aim of the study was to determine whether or not the combined use of the activity of ADA, its isoenzymes and differential cell counts would provide a more efficient means of diagnosing tuberculosis than the use of ADA levels alone. Results: Data suggested significant (p0.75) of ADA/sub 2/ADA was found to be better indicator of tuberculosis. Lymphocyte neutrophil ratio (L/N)> 0.69 gave additional benefit to increase the sensitivity and specificity for the use of ADA as marker in diagnosing tuberculosis. Conclusion: The combined use of activity of ADA, its isoenzymes and total and differential cell counts is a better indicator and gives better understanding to diagnose and evaluate tuberculosis and response to therapy. (author)

  4. Serum Adenosine deaminase activity and C-reactive protein levels in unstable angina

    Rani Surekha

    2003-01-01

    Full Text Available In unstable angina (USA patients, immunological responses contributing to inflammation play a vital role in plaque rupture and thrombosis causing stroke. In the present study an attempt is made to estimate the levels of adenosine deaminase activity, an immunoenzyme marker and C-reactive protein, a marker of inflammation in USA patients. 45 patients presenting USA and 50 age and sex matched healthy controls were included in the study. Serum ADA activity was measured spectrophotometrically at 630nm and serum C-reactive protein was detected using Avitex CRP kit, which is a rapid latex agglutination test. The Mean ADA levels were 41.15 ± 11.04 in patients and 20.71±5.63 in controls and 66.6% of patients and none of the controls were positive to CRP. The present study observed the importance of ADA as a serum marker in addition to CRP for assessing the immune response in USA patients.

  5. Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

    Taori Girdhar M

    2006-03-01

    Full Text Available Abstract Background Tuberculous meningitis (TBM is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB. The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA activity in cerebrospinal fluid (CSF of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94, mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96 and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68, P Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

  6. Purine metabolic enzymes in lymphocytes. III. Effects of immunosuppressants on adenosine deaminase and purine nucleoside phosphorylase activities

    Kurashige, Satonori; Akuzawa, Yuki; Yoshida, Toshiharu; Kodama, Kazue

    1983-01-01

    Mice were treated with a single injection of 6-mercaptopurine riboside (6MP-R), predonine or cyclophosphamide (CY), and the effects of these immunosuppressants on blastogenic responses to phytohemagglutinin P (PHA-P) or bacterial lipopoly saccharide (LPS) and on adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) activities were studied with spleen lymphocytes. The retardation of blastogenic responses to both PHA-P and LPS were associated with the retardation of both intracellu...

  7. Comparative study of adenosine deaminase activity, insulin resistance and lipoprotein(a) among smokers and healthy non-smokers

    Ramesh Ramasamy; Sathish Babu Murugaiyan; Arulkumaran U.; Sathiya R.; Kuzhandai Velu V.; Niranjan Gopal

    2016-01-01

    Background: Adenosine deaminase also known as adenosine aminohydrolase involved in purine metabolism. Its primary function is development and maintenance of immune system. The main objective of the study was to estimate adenosine deaminase (ADA) enzyme and find its correlation with lipoprotein(a) and insulin resistance among smokers and healthy non-smokers. Methods: Fifty smokers and fifty healthy non-smokers were selected based on WHO definition. ADA, lipid profile and glucose was estimat...

  8. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

    Iván Darío BRAVO-TOBAR

    2015-10-01

    Full Text Available SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA and C-reactive protein serum levels (CRP in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35, II (n = 29, and III (n = 18. A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

  9. Non-linear quantitative structure-activity relationship for adenine derivatives as competitive inhibitors of adenosine deaminase

    Logistic regression and artificial neural networks have been developed as two non-linear models to establish quantitative structure-activity relationships between structural descriptors and biochemical activity of adenosine based competitive inhibitors, toward adenosine deaminase. The training set included 24 compounds with known k i values. The models were trained to solve two-class problems. Unlike the previous work in which multiple linear regression was used, the highest of positive charge on the molecules was recognized to be in close relation with their inhibition activity, while the electric charge on atom N1 of adenosine was found to be a poor descriptor. Consequently, the previously developed equation was improved and the newly formed one could predict the class of 91.66% of compounds correctly. Also optimized 2-3-1 and 3-4-1 neural networks could increase this rate to 95.83%

  10. Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

    Márcio Roberto Silva

    2006-06-01

    Full Text Available Determination of seric levels of adenosine deaminase (ADA, an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB. In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated, and TB-negative animals (n = 204, TB-free herds. The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L was significantly lower (p = 0.008 than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L. When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 ± 3.75 U/L was not significantly different anymore from that of the TB-positive group (p = 0.28. There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively. In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

  11. Aqueous extract from taxus baccata inhibits adenosine deaminase activity significantly in cancerous and noncancerous human gastric and colon tissues

    Zahide Esra Durak

    2014-01-01

    Full Text Available Objective: To investigate possible effects of aqueous taxus baccata extract on adenosine deaminase (ADA activity in cancerous and noncancerous human tissues and to clarify mechanism(s of its anticancer potential. Materials and Methods: Cancerous and noncancerous human gastric and colon tissues were used in the study. The extracts were prepared in distilled water. Before and after treatment with the extracts, ADA activities in the tissue homogenates were measured. Results: ADA activity was found to be higher in gastric tissue compared with colon tissue, but no differences were found between ADA activities of cancerous and noncancerous tissues for both as well. In the plant extract studies, it was found that taxus extract significantly inhibited ADA activity both in cancerous and noncancerous gastric and colon tissues. Conclusion: Our results suggest that aqueous extract from taxus baccata inhibits ADA activities in both gastric and colon tissues significantly. It is suggested that in addition to other proposed mechanisms, accumulated adenosine due to the inhibition of ADA enzyme might also play part in the anticancer properties of taxus species.

  12. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment. PMID:26769247

  13. Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

    Akinyemi, Ayodele Jacob; Thomé, Gustavo Roberto; Morsch, Vera Maria; Bottari, Nathieli B; Baldissarelli, Jucimara; de Oliveira, Lizielle Souza; Goularte, Jeferson Ferraz; Belló-Klein, Adriane; Oboh, Ganiyu; Schetinger, Maria Rosa Chitolina

    2016-07-01

    Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27151061

  14. Mixed Inhibition of Adenosine Deaminase Activity by 1,3-Dinitrobenzene: A Model for Understanding Cell-Selective Neurotoxicity in Chemically-Induced Energy Deprivation Syndromes in Brain

    Wang, Yipei; Liu, Xin; Schneider, Brandon; Zverina, Elaina A.; Russ, Kristen; Wijeyesakere, Sanjeeva J.; Fierke, Carol A.; Richardson., Rudy J.; Philbert, Martin A.

    2011-01-01

    Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ...

  15. Study of Ectonucleotidases and Adenosine Deaminases in Drosophila

    PREUER, Kristina

    2013-01-01

    Extracellular adenosine triphosphate and extracellular adenosine are important regulatory molecules in the human immune system. The concentrations of these molecules are in turn regulated by ectonucleotidases and adenosine deaminases. In this thesis I attempt to test the gene silencing efficiency of RNA interference for three different genes coding for such enzymes in the model organism Drosophila melanogaster.

  16. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    ... links) CLIMB: Children Living with Inherited Metabolic Diseases Muscular Dystrophy Association: Myoadenylate Deaminase Deficiency Genetic Testing Registry (1 link) Muscle AMP deaminase deficiency ...

  17. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2) in schizophrenic patients Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2) em pacientes esquizofrênicos

    Gustavo Pimentel Dutra; Gustavo L. Ottoni; Lara, Diogo R; Maurício Reis Bogo

    2010-01-01

    OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A) (ADA1*2) exhibits 20-30% lower e...

  18. A CORRELATIVE STUDY OF ADENOSINE DEAMINASE ACTIVITY & T.B. IgG IN SERUM IN CASES OF TUBERCULOSIS

    Ajay

    2012-11-01

    Full Text Available ABSTRACT: INTRODUCTION: Tuberculosis is major cause of morbidity and mort ality in India as well in other parts of world. It is caused by myc obacterium tuberculosis which primarily affects lung and cause pulmonary tuberculosis. Diag nosis of tuberculosis rests upon a positive history of contact, clinical symptoms, x-ray chest, sputum positivity and AFB culture. Adenosine deaminase (ADA is an enzyme which catalyzes the de amination of adenosine into inosine and ammonia. ADA level is found to be elevated in tuber culosis and typhoid fever where cell mediated immunity is elevated. The ADA level is sig nificantly elevated in tuberculosis and helps to differentiate between tubercular and non tubercu lar diseases. The ADA level is also found to be elevated in serum and pleural fluid in patients of tubercular pleural effusion, tubercular ascitis and tubercular pericardial effusion. METHODS : Routine hemogram, Montoux test, X-ray chest, FNAC of lymph nodes, biopsy of lymph node whene ver required, estimation of serum ADA level and T.B.IgG studies were performed in each cas e. RESULTS: In the present study a total of 45 cases were selected for the study. There are 30 cases of pulmonary tuberculosis and 15 controls. The values of serum ADA and tubercular Ig G in pulmonary tubercular group are significantly higher as compared to those of control s. None of the control for ADA showed significant ratio of positivity (≥1.7. One of the 1 5 cases showed remarkable ratio of positivity (>1.2-1.6 and 14 (93.3% cases showed insignifican t ratio of positivity. Only 2 (13.33% of the 15 cases showed positivity for TB IgG and rest 13 (8 6.66% were regarded negative. CONCLUSIONS: Thus it can be concluded that determination of ser um adenosine deaminase levels can effectively diagnose tuberculosis with s ensitivity of 96.66% and specificity of 93.33% as compared to TBIgG showing sensitivity of 90% and specificity of 86.6%. Also cost of ADA estimation is remarkably

  19. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.)

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 μM adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors)

  20. The emerging role of adenosine deaminases in insects

    Doleželová, Eva; Žurovec, Michal; Doležal, T.; Šimek, Petr; Bryant, P. J.

    2005-01-01

    Roč. 35, č. 5 (2005), s. 381-389. ISSN 0965-1748 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant ostatní: United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : adenosine deaminase * ADA * growth factor Subject RIV: ED - Physiology Impact factor: 2.733, year: 2005

  1. Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.

    Baldissera, Matheus D; Bottari, Nathieli B; Mendes, Ricardo E; Schwertz, Claiton I; Lucca, Neuber J; Dalenogare, Diessica; Bochi, Guilherme V; Moresco, Rafael N; Morsch, Vera M; Schetinger, Maria R C; Rech, Virginia C; Jaques, Jeandre A; Da Silva, Aleksandro S

    2015-11-01

    The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease. PMID:26452485

  2. Ectonucleotidases and adenosine deaminase activity in laying hens naturally infected by Salmonella Gallinarum and their effects on the pathogenesis of the disease.

    Boiago, Marcel M; Baldissera, Matheus D; Doleski, Pedro H; Bottari, Nathieli B; do Carmo, Guilherme M; Araujo, Denise N; Giuriatti, Jessica; Baggio, Vanessa; Leal, Daniela B R; Casagrande, Renata A; Wisser, Cláudia S; Stefani, Lenita M; da Silva, Aleksandro S

    2016-04-01

    Salmonella Gallinarum is the etiologic agent of fowl typhoid that affects chickens and turkeys causing egg production drops, infertility, lower hatchability, high mortality, and as a consequence severe economic losses to the poultry industry. The alterations in NTPDase and adenosine deaminase (ADA) activities have been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this infection. Thus, the aim of this study was to evaluate the activities of NTPDase, 5'nucleotidase, and ADA in serum and hepatic tissue of laying hens naturally infected by Salmonella Gallinarum. Liver and serum samples were collected of 27 laying hens (20 S. Gallinarum infected and 7 uninfected). NTPDase and 5'-nucleotidase activities in serum were increased (P laying hens naturally infected by S. Gallinarum; as well as increased (P laying hens. Histopathological analyses revealed that S. Gallinarum caused fibrinoid necrosis in liver and spleen associated with infiltrates of heterophils, macrophages, lymphocytes, and plasma cells. Considering that NTPDase and ADA are involved in the cell-mediated immunity, this study suggests that activities of these enzymes could be important biomarkers to determine the severity of inflammatory and immune responses in salmonellosis, contributing to clarify the pathogenesis of the disease. PMID:26911648

  3. Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

    Liang, Dongchun; Zuo, Aijun; Zhao, Ronglan; Shao, Hui; Kaplan, Henry J; Sun, Deming

    2016-03-15

    Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies. PMID:26856700

  4. Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjusated polymers

    HE Fang; YU MingHui; WANG Shu

    2009-01-01

    A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deami-nase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine (biotin-aptamer),a signaling probe single-stranded DNA-tagged fiuorescein at terminus (ssDNA-FI) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized,and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA (biotin-dsDNA-FI).The ssONA-FI is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.

  5. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.); Determination radiochromatographique de l'adenosine deaminase (A.D.)

    Chivot, J.J.; Depernet, D.; Caen, J. [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes

    1970-07-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 {mu}M adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [French] Nous avons pu, en utilisant une methode radiochromatographique, mesurer une activite adenosine deaminasique dans le plasma humain pauvre en plaquettes heparine qui peut degrader 0,016 {mu}M d'adenosine. Cette activite qui est supprimee par chauffage a 56 degres pendant 30 minutes, est reduite par conservation a -20 C pendant une semaine, est inhibee par d'importantes concentrations d'uree et ne l'est pas, ni par le dipyridamol, ni par le pHMB. Cette activite est proportionnelle a la quantite de plasma, source d'enzyme, mise dans les differents systemes reactifs. (auteur)

  6. Serum adenosine deaminase as oxidative stress marker in type 2 diabetes mellitus

    Shashikala Magadi Dasegowda

    2015-05-01

    Results: The study observed an increased level of serum adenosine deaminase, malondialdehyde and decreased levels of total antioxidant capacity in type 2 diabetes mellitus compared to controls. Serum adenosine deaminase levels in type 2 diabetics were 50.77 +/- 6.95 and in controls was 17.86 +/- 4.04. Serum Malondialdehyde levels in type 2 diabetics was 512.13 +/- 70.15 and in controls was 239.32 +/- 23.97. Serum total antioxidant levels in type 2 diabetics was 0.39+/-0.15 and in controls was 1.66+/-0.25. Positive correlation was seen between serum adenosine deaminase and malondialdehyde and it was statistically significant. Statistically significant negative correlation was seen between serum adenosine deaminase and total antioxidant capacity. Conclusion: Adenosine deaminase can be used as oxidative stress marker. Their increased levels indicate oxidative stress in type 2 diabetes mellitus. Therefore, estimation of serum adenosine deaminase levels help in early prediction and prevention of long term complications occurring due to oxidative stress in diabetics, thereby decreasing the mortality and morbidity in them. [Int J Res Med Sci 2015; 3(5.000: 1195-1198

  7. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  8. SIGNIFICANCE OF ADENOSINE DEAMINASE SERUM CONCENTRATIONS IN THE DIAGNOSIS OF EXTRA-PULMONARY TUBERCULOSIS

    Stevanovic G,

    2011-06-01

    Full Text Available Extra pulmonary tuberculosis (EPTB is a growing problem worldwide. Due to the nature of the disease, the diversity of clinical pictures as well as its minor epidemiological importance, the diagnosis is difficult and often late.In addition to standard TB diagnostic techniques use of new biochemical (surrogate markers are increased. With this work we wanted to examine the usefulness of serum adenosine deaminase levels as a diagnostic parameter for EPTB.The work included 116 patients with fever of unknown origin in which tuberculosis or infectious mononucleosis was not proven and 51 person who had proven EPTB. Correlated adenosine deaminase levels between these two groups we obtained significantly higher values ​​in patients with EPTB. The calculated sensitivity was 0.56, specificity 0.89, positive predictive value 0.80 and negative predictive value 0.72. Certain reducing of the values observed during anti TB therapy. In previous studies the diagnostic importance of adenosine deaminase in the diagnosis of tuberculosis serosityes was demonstrated. The significance of serum levels in diagnosis is rarely evaluated during EPTB. Our findings are similar to the results of authors who have conducted such testing in the pediatric population.Increased concentrations of serum adenosine deaminase have shown the potential of usable screening test and can be used as an indicative EPTB parameter. To fully assess its diagnostic significance require future clinical research.

  9. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency

    Hassan, Amel; Booth, Claire; Brightwell, Alex; Allwood, Zoe; Veys, Paul; Rao, Kanchan; Hoenig, Manfred; Friedrich, Wilhelm; Gennery, Andrew; Slatter, Mary; Bredius, Robbert; Finocchi, Andrea; Cancrini, Caterina; Aiuti, Alessandro; Porta, Fulvio; Lanfranchi, Arnalda; Ridella, Michela; Steward, Colin; Filipovich, Alexandra; Marsh, Rebecca; Bordon, Victoria; Al-Muhsen, Saleh; Al-Mousa, Hamoud; Alsum, Zobaida; Al-Dhekri, Hasan; Al Ghonaium, Abdulaziz; Speckmann, Carsten; Fischer, Alain; Mahlaoui, Nizar; Nichols, Kim E.; Grunebaum, Eyal; Al Zahrani, Daifulah; Roifman, Chaim M.; Boelens, Jaap; Davies, E. Graham; Cavazzana-Calvo, Marina; Notarangelo, Luigi; Gaspar, H. Bobby

    2012-01-01

    Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed o

  10. Investigation into effects of antipsychotics on ectonucleotidase and adenosine deaminase in zebrafish brain.

    Seibt, Kelly Juliana; Oliveira, Renata da Luz; Bogo, Mauricio Reis; Senger, Mario Roberto; Bonan, Carla Denise

    2015-12-01

    Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug. PMID:26156500

  11. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

    Medin, J A; Hunt, L; Gathy, K; Evans, R K; Coleman, M S

    1990-01-01

    Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellul...

  12. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA)-deficiency

    Aisha Vanessa Sauer; Immacolata eBrigida; Nicola eCarriglio; Alessandro eAiuti

    2012-01-01

    Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA) or hematopoietic stem cell gene therapy (HSC-GT). Although ...

  13. Serum adenosine deaminase as oxidative stress marker in type 2 diabetes mellitus

    Shashikala Magadi Dasegowda; Ashok Kumar Jeppu; Sushith; Kavitha Ashok Kumar

    2015-01-01

    Background: Oxidative stress markers are increased in type 2 diabetes mellitus and its estimation helps in predicting the long term complications. In present study comparison and correlation of the levels of serum adenosine deaminase, serum malondialdehyde, and serum total antioxidant capacity in type 2 diabetes mellitus and in age and sex matched healthy controls. Methods: Study group consisted of 100 individuals between the age group of 35-65 years of age. Of which 50 individuals with t...

  14. Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency.

    Montiel-Equihua, Claudia A; Thrasher, Adrian J.; Bobby Gaspar, H

    2009-01-01

    Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID) and especially adenosine deaminase (ADA)-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a pr...

  15. Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency

    Thrasher, Adrian

    2009-01-01

    Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID) and especially adenosine deaminase (ADA)-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a pr...

  16. SIGNIFICANCE OF ADENOSINE DEAMINASE SERUM CONCENTRATIONS IN THE DIAGNOSIS OF EXTRA-PULMONARY TUBERCULOSIS

    Stevanovic G.; Pelemis M.; Pavlovic M.; Lavadinovic L,; Dakic Z,; Milosevic I,; Milosevic B.

    2011-01-01

    Extra pulmonary tuberculosis (EPTB) is a growing problem worldwide. Due to the nature of the disease, the diversity of clinical pictures as well as its minor epidemiological importance, the diagnosis is difficult and often late.In addition to standard TB diagnostic techniques use of new biochemical (surrogate markers) are increased. With this work we wanted to examine the usefulness of serum adenosine deaminase levels as a diagnostic parameter for EPTB.The work included 116 patients with feve...

  17. Adenosine deaminase regulates Treg expression in autologous T cell-dendritic cell cocultures from patients infected with HIV-1.

    Naval-Macabuhay, Isaac; Casanova, Víctor; Navarro, Gemma; García, Felipe; León, Agathe; Miralles, Laia; Rovira, Cristina; Martinez-Navio, José M; Gallart, Teresa; Mallol, Josefa; Gatell, José M; Lluís, Carme; Franco, Rafael; McCormick, Peter J; Climent, Núria

    2016-02-01

    Regulatory T cells have an important role in immune suppression during HIV-1 infection. As regulatory T cells produce the immunomodulatory molecule adenosine, our aim here was to assess the potential of adenosine removal to revert the suppression of anti-HIV responses exerted by regulatory T cells. The experimental setup consisted of ex vivo cocultures of T and dendritic cells, to which adenosine deaminase, an enzyme that hydrolyzes adenosine, was added. In cells from healthy individuals, adenosine hydrolysis decreased CD4(+)CD25(hi) regulatory T cells. Addition of 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, significantly decreased CD4(+)CD25(lo) cells, confirming a modulatory role of adenosine acting via adenosine receptors. In autologous cocultures of T cells with HIV-1-pulsed dendritic cells, addition of adenosine deaminase led to a significant decrease of HIV-1-induced CD4(+)CD25(hi) forkhead box p3(+) cells and to a significant enhancement of the HIV-1-specific CD4(+) responder T cells. An increase in the effector response was confirmed by the enhanced production of CD4(+) and CD8(+) CD25(-)CD45RO(+) memory cell generation and secretion of Th1 cytokines, including IFN-γ and IL-15 and chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5. These ex vivo results show, in a physiologically relevant model, that adenosine deaminase is able to enhance HIV-1 effector responses markedly. The possibility to revert regulatory T cell-mediated inhibition of immune responses by use of adenosine deaminase, an enzyme that hydrolyzes adenosine, merits attention for restoring T lymphocyte function in HIV-1 infection. PMID:26310829

  18. Mitochondrial Damage and Apoptosis Induced by Adenosine Deaminase Inhibition and Deoxyadenosine in Human Neuroblastoma Cell Lines.

    Garcia-Gil, Mercedes; Tozzi, Maria Grazia; Balestri, Francesco; Colombaioni, Laura; Camici, Marcella

    2016-07-01

    The treatment with deoxycoformycin, a strong adenosine deaminase inhibitor, in combination with deoxyadenosine, causes apoptotic cell death of two human neuroblastoma cell lines, SH-SY5Y and LAN5. Herein we demonstrate that, in SH-SY5Y cells, this combination rapidly decreases mitochondrial reactive oxygen species and, in parallel, increases mitochondrial mass, while, later, induces nuclear fragmentation, and activation of caspase-8, -9, and -3. In previous papers we have shown that a human astrocytoma cell line, subjected to the same treatment, undergoes apoptotic death as well. Therefore, both astrocytoma and neuroblastoma cell lines undergo apoptotic death following the combined treatment with deoxycoformycin and deoxyadenosine, but several differences have been found in the mode of action, possibly reflecting a different functional and metabolic profile of the two cell lines. Overall this work indicates that the neuroblastoma cell lines, like the line of astrocytic origin, are very sensitive to purine metabolism perturbation thus suggesting new therapeutic approaches to nervous system tumors. J. Cell. Biochem. 117: 1671-1679, 2016. © 2015 Wiley Periodicals, Inc. PMID:26659614

  19. Hereditary overexpression of adenosine deaminase in erythrocytes: Evidence for a cis-acting mutation

    Chen, E.H. (Univ. of Michigan, Ann Arbor, MI (United States)); Tartaglia, A.P. (Albany Medical College, Albany, MI (United States)); Mitchell, B.S. (Univ. of North Carolina, Chapel Hill, NC (United States))

    1993-10-01

    Overexpression of adenosine deaminase (ADA) in red blood cells is inherited as an autosomal dominant trait and causes hemolytic anemia. The increased ADA activity in erythrocytes is due to an increase in steady-state levels of ADA mRNA of normal sequence. Increased ADA mRNA may be due to a cis-acting mutation which results in increased transcription or a loss of down-regulation during erythroid differentiation. Alternatively, it is possible that the mutation is in a trans-acting factor which interacts with normal ADA transcriptional elements to cause overexpression in red blood cells. To discriminate between a cis-acting and a trans-acting mutation, the authors took advantage of a highly polymorphic TAAA repeat located at the tail end of an Alu repeat approximately 1.1 kb upstream of the ADA gene. Using PCR to amplify this region, the authors identified five different alleles in 19 members of the family. All 11 affected individuals had an ADA allele with 12 TAAA repeats, whereas none of the 8 normal individuals did. The authors conclude that this disorder results from a cis-acting mutation in the vicinity of the ADA gene. 24 refs., 3 figs.

  20. The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

    Bergamaschi Antonio

    2008-09-01

    Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

  1. Oxidative Stress Biomarkers and Adenosine Deaminase over the Alopecic Area of the Patients with Alopecia Areata

    Öztürk, Perihan; Arıcan, Özer; Kurutaş, Ergül Belge; Mülayim, Kamil

    2016-01-01

    Background: Alopecia areata (AA) is an autoimmune, T-cell mediated, and chronic inflammatory disorder. The pathological mechanisms of disease are unclear, but oxidative stress may be involved. To our knowledge, no studies have examined the oxidative stress levels or biomarkers within the lesional area and skin surface in patients with AA. Similarly, adenosine deaminase (ADA) has not been characterized in AA. Aims: Therefore, we aimed to define ADA levels and the factors involved in oxidative stress from scalp-scrapes of patients with AA. Study Design: Case-control study. Method: A total of 60 patients (30 diagnosed AA patients and 30 healthy controls) were included in the study. ADA as well as oxidative stress factors, including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were analyzed from scalp-scrapes in both groups and quantified by spectrophotometry. Results: Activities of SOD (p=0.000), CAT (p=0.033), and ADA (p=0.004) as well as levels of GSH (p=0.000) and MDA (p=0.032) in patients with AA were higher than the controls statistically significant. Conclusion: Based on these results, factors associated with oxidative stress were elevated in AA patient scalp-scrapes compared to controls and may have a defined role the disease pathogenesis. Alterations in the activities of antioxidant enzymes from AA patient scraping samples may be a local effect of elevated oxidative stress levels. In this disease, oxidative stress may affect not only hair follicle but also any layers of the skin.

  2. A 24-Year Enzyme Replacement Therapy in an Adenosine-deaminase-Deficient Patient.

    Tartibi, Hana M; Hershfield, Michael S; Bahna, Sami L

    2016-01-01

    Severe combined immunodeficiency (SCID) is a fatal childhood disease unless immune reconstitution is performed early in life, with either hematopoietic stem cell transplantation or gene therapy. One of its subtypes is caused by adenosine deaminase (ADA) enzyme deficiency, which leads to the accumulation of toxic metabolites that impair lymphocyte development and function. With the development of polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) enzyme replacement therapy, many ADA-deficient children with SCID who could not receive a hematopoietic stem cell transplantation or gene therapy survived and had longer and healthier lives. We report a 24-year course of treatment in a patient who was diagnosed with ADA deficiency at 4 months of age. The patient was treated with PEG-ADA, which was the only therapy available for him. The patient's plasma ADA level was regularly monitored and the PEG-ADA dose adjusted accordingly. This treatment has resulted in near-normalization of lymphocyte counts, and his clinical course has been associated with only minor to moderate infections. Thus far, he has had no manifestations of autoimmune or lymphoproliferative disorders. This patient is among the longest to be maintained on PEG-ADA enzyme replacement therapy. PMID:26684479

  3. Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency

    Claudia A Montiel-Equihua

    2009-12-01

    Full Text Available Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID and especially adenosine deaminase (ADA-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a protocol for the autologous transplant of retroviral vector-mediated gene-modified hematopoietic stem cells, which has proved to be both successful and, to date, safe. Patients in trials in three different countries have shown long-term immunological and metabolic correction. Nevertheless, improvements to the safety profile of viral vectors are underway and will undoubtedly reinforce the position of stem cell gene therapy as a treatment option for ADA-SCID.Keywords: adenosine deaminase, severe combined immunodeficiency, gene therapy, hematopoietic stem cell, retrovirus, clinical trial

  4. Dosagem sérica de adenosina deaminase em lúpus eritematoso sistêmico: ausência de associação com atividade de doença Levels of serum adenosine deaminase in systemic lupus erythematosus: lack of association with disease activity

    Isabella Lima

    2005-10-01

    Full Text Available O lúpus eritematoso sistêmico (LES é uma doença inflamatória auto-imune, que evolui intercalando períodos de atividade e remissão. OBJETIVO: avaliar a associação da dosagem sérica de adenosina deaminase (ADA e atividade do LES, segundo os critérios do SLEDAI 2K - Systemic lupus erythematosus disease activity index. MÉTODOS: avaliou-se 82 pacientes com LES atendidos em um hospital de referência para o tratamento do LES em Salvador, BA, Brasil. A atividade de doença foi determinada pelo SLEDAI 2K e a dosagem sérica da ADA realizada por colorimetria. RESULTADOS: oitenta e uma pacientes (98,78% eram do sexo feminino e a idade média foi de 35,07±11,73 anos. O escore de SLEDAI médio foi de 11,66±5,89; a média de ADA sérica foi de 38,24±13,61U/l; C3 de 91,93±27,39 mg/dl; C4 de 15,17±5,77 mg/dl e a pesquisa de anticorpos anti-DNA nativo (aDNAn foi positiva em 31 casos (37,8%. Não houve correlação entre os níveis séricos de ADA e escore do SLEDAI. A ADA sérica correlacionou-se inversamente com C4 (r=-0,336 e p=0,001. CONCLUSÕES: no presente estudo a dosagem sérica de ADA não se associou a atividade de doença segundo os critérios do SLEDAI 2K, sugerindo que esse teste não deve ser utilizado como marcador de atividade de doença em LES. Esse resultado diverge da maioria dos trabalhos publicados, o que pode ser explicado pela dificuldade de padronização da técnica de dosagem da ADA ou por diferença nas diversas populações estudadas.Systemic lupus erythematosus (SLE is an autoimmune inflammatory disease, with a variable course and characterized by periods of remissions and exacerbations. OBJECTIVE: To evaluate the association between serum adenosine deaminase (ADA levels and disease activity in SLE. METHODS: Eighty two SLE patients seen at Santa Izabel Hospital in Salvador, BA, Brazil, were studied. Disease activity was measured by SLEDAI 2K- Systemic Lupus Erythematosus Disease Activity Index, and serum ADA was

  5. Comparison of interferon-gamma release assays and adenosine deaminase of pleural fluid for the diagnosis of pleural tuberculosis

    刘菲

    2014-01-01

    Objective To compare the diagnostic performance of interferon gamma releasing assays(T-SPOT.TB)and adenosine deaminase(ADA)in pleural tuberculosis,and therefore to evaluate the value of T-SPOT.TB in a high tuberculosis burden country.Methods From June 2011to November 2012,111 patients with pleural fluid in Beijing Chest Hospital,Capital Medical University were

  6. MicroRNA-146b-3p Regulates Retinal Inflammation by Suppressing Adenosine Deaminase-2 in Diabetes

    Sadanand Fulzele

    2015-01-01

    Full Text Available Hyperglycemia- (HG- Amadori-glycated albumin- (AGA- induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR. There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3′-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3′-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-α release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2.

  7. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2 in schizophrenic patients Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2 em pacientes esquizofrênicos

    Gustavo Pimentel Dutra

    2010-09-01

    Full Text Available OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A (ADA1*2 exhibits 20-30% lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2 in schizophrenic patients and healthy controls. METHOD: The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. RESULTS: A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6% relative to controls (13/111 - 11.7%, p = 0.032, OR = 2.6. CONCLUSION: These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.OBJETIVO: A adenosina pode ter um papel importante na fisiopatologia da esquizofrenia, uma vez que modula a liberação de vários neurotransmissores, tais como glutamato, dopamina, serotonina e acetilcolina, diminui a atividade neuronal por hiperpolarização pós-sináptica e inibe a atividade dopaminérgica. A adenosina desaminase participa do metabolismo das purinas pela conversão de adenosina em inosina. O mais frequente polimorfismo funcional da adenosina desaminase (22G →A (ADA1*2 exibe uma diminuição de 20-30% da atividade funcional em indivíduos com genótipo G/A quando comparados com indivíduos com o genótipo G/G. O objetivo deste estudo foi avaliar o polimorfismo 22G→A (ADA1*2 em pacientes esquizofrênicos e em

  8. The value of adenosine deaminase, interferon-gamma, and interferon-gamma induced protein of 10kD in the diagnosis of tuberculous pleuritis

    Ya-kun DONG; Li, Ai-zhen; Li-heng ZHENG; Yue-peng CHI; Wang, Yu-Hong; Qiu-mei WANG; Zhu, Gui-Yun; Lan-pin XIE

    2015-01-01

    Objective To explore the value of adenosine deaminase (ADA) activity, interferon-gamma (IFN-γ) and IFN-γ induced protein of 10kD (IP-10) levels in pleural effusion for the diagnosis of tuberculous pleuritis. Methods ADA activity, IFN-γ and IP-10 levels in pleural effusion were determined in sixty-three patients with tuberculous pleuritis and 50 patients with malignant pleural effusion. Results The mean levels of ADA, IFN-γ and IP-10 in the tuberculous pleural effusion were significantly highe...

  9. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn2+ ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P42212, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (VM) of 3.3 Å3 Da−1 and a solvent content of 62.7%

  10. Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo.

    Lim, B; Williams, D A; Orkin, S H

    1987-01-01

    Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free r...

  11. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  12. Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency.

    Hassan, Amel; Booth, Claire; Brightwell, Alex; Allwood, Zoe; Veys, Paul; Rao, Kanchan; Hönig, Manfred; Friedrich, Wilhelm; Gennery, Andrew; Slatter, Mary; Bredius, Robbert; Finocchi, Andrea; Cancrini, Caterina; Aiuti, Alessandro; Porta, Fulvio; Lanfranchi, Arnalda; Ridella, Michela; Steward, Colin; Filipovich, Alexandra; Marsh, Rebecca; Bordon, Victoria; Al-Muhsen, Saleh; Al-Mousa, Hamoud; Alsum, Zobaida; Al-Dhekri, Hasan; Al Ghonaium, Abdulaziz; Speckmann, Carsten; Fischer, Alain; Mahlaoui, Nizar; Nichols, Kim E; Grunebaum, Eyal; Al Zahrani, Daifulah; Roifman, Chaim M; Boelens, Jaap; Davies, E Graham; Cavazzana-Calvo, Marina; Notarangelo, Luigi; Gaspar, H Bobby

    2012-10-25

    Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed outcome of HCT in 106 patients with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family donors (MSDs, MFDs) had significantly better overall survival (86% and 81%) in comparison with HCT from matched unrelated (66%; P HCT, nonengraftment was a major problem. Long-term immune recovery showed that regardless of transplant type, overall T-cell numbers were similar, although a faster rate of T-cell recovery was observed after MSD/MFD HCT. Humoral immunity and donor B-cell engraftment was achieved in nearly all evaluable surviving patients and was seen even after unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and show that, if patients survive HCT, long-term cellular and humoral immune recovery is achieved. PMID:22791287

  13. STUDY OF ADENOSINE DEAMINASE AND LYMPHOCYTE/NEUTROPHIL RATIO IN COMBINATION AS DIAGNOSTIC TOOL FOR TUBERCULAR PLEURAL EFFUSION

    Md. Faizur

    2015-10-01

    Full Text Available Adenosine deaminase, considered one of the key enzyme of purine metabolism, has been used in work up of lymphocytic pleural effusion. Low level of ADA 50IU/L specially when combined with Lymphocytic/neutrophil ratio >0.75 in pleural fluid is useful test in the diagnosis of tubercular pleurisy. AIM AND OBJECTIVE: To suggest a better diagnostic tool in the diagnosis of pleural effusion of tubercular origin by estimating the activity of ADA along with L/N ratio in pleural effusion. METHOD: Biochemical, cytological and microbiology studies were done by obtaining pleural fluid by thoracocentesis in 100 patients after excluding pleural effusion cases of malignancy, transudative effusion. RESULT: 84 cases were tubercular and had high level of ADA in comparison to rest of 16 non-tubercular cases. At level of 50 IU/L of ADA activity test had sensitivity of 97.6%, specificity 87.5%, positive predictive value 97.6%, negative predictive value 87.5%which increased to 100% and 92.8%, 98.6%, and 100% respectively in combination with test of Lymphocytic/Neutrophilic ratio >0.75. CONCLUSION: ADA level with L/N ratio can be important investigation in diagnosis of tubercular pleural effusion cases.

  14. Increased level of soluble adenosine deaminase in bone marrow of visceral leishmaniasis patients: an inverse relation with parasite load.

    Rai, Ambak K; Kumar, Prabin; Saini, Sheetal; Thakur, Chandreshwar P; Seth, Tulika; Mitra, Dipendra K

    2016-09-01

    Adenosine deaminase (ADA) which degrades adenosine to inosine, is known to be pro-inflammatory molecule in many diseases. Adenosine suppresses the functioning of the immune system and thus promotes dissemination of the parasite. In our previous finding, the level of soluble ADA in serum of visceral leishmaniasis (VL) was found to be increased as compared to healthy controls. However, it cannot be fairly interpreted unless their level is demonstrated at the disease site, where the parasite resides. We designed this study to correlate the level of soluble ADA (sADA) with parasitic load at the disease site i.e. bone marrow (BM). We found increased levels of sADA in BM as compared to the unaffected BM. Furthermore, a significant inverse correlation is observed between the parasite load and level of sADA at the disease site. PMID:27447233

  15. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA-deficiency

    Aisha Vanessa Sauer

    2012-08-01

    Full Text Available Genetic defects in the adenosine deaminase (ADA gene are among the most common causes for severe combined immunodeficiency (SCID. ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT, enzyme replacement therapy with bovine ADA (PEG-ADA or hematopoietic stem cell gene therapy (HSC-GT. Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment.A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T and B cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.

  16. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  17. Inhibition of adenosine deaminase (ADA)-mediated metabolism of cordycepin by natural substances

    Li, Gen; Nakagome, Izumi; Hirono, Shuichi; Itoh, Tomoo; Fujiwara, Ryoichi

    2015-01-01

    Cordycepin, which is an analogue of a nucleoside adenosine, exhibits a wide variety of pharmacological activities including anticancer effects. In this study, ADA1- and ADA2-expressing HEK293 cells were established to determine the major ADA isoform responsible for the deamination of cordycepin. While the metabolic rate of cordycepin deamination was similar between ADA2-expressing and Mock cells, extensive metabolism of cordycepin was observed in the ADA1-expressing cells with K m and V max v...

  18. Activation induced deaminase: how much and where?

    Orthwein, Alexandre; Di Noia, Javier M

    2012-08-01

    Activation induced deaminase (AID) plays a central role in adaptive immunity by initiating the processes of somatic hypermutation (SHM) and class switch recombination (CSR). On the other hand, AID also predisposes to lymphoma and plays a role in some autoimmune diseases, for which reasons AID expression and activity are regulated at various levels. Post-translational mechanisms regulating the amount and subcellular localization of AID are prominent in balancing AID physiological and pathological functions in B cells. Mechanisms regulating AID protein levels include stabilizing chaperones in the cytoplasm and proteins efficiently targeting AID to the proteasome within the nucleus. Nuclear export and cytoplasmic retention contribute to limit the amount of AID accessing the genome. Additionally, a number of factors have been implicated in AID active nuclear import. We review these intertwined mechanisms proposing two scenarios in which they could interact as a network or as a cycle for defining the optimal amount of AID protein. We also comparatively review the expression levels of AID necessary for its function during the immune response, present in different cancers as well as in those tissues in which AID has been implicated in epigenetic remodeling of the genome by demethylating DNA. PMID:22687198

  19. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    Ku, Min-Je [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Won-Ho [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Nam, Ki-hyun; Rhee, Kyeong-hee [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Ki-Seog [Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Eunice EunKyung [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Yu, Myung-Hee [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Hwang, Kwang Yeon, E-mail: hwangky@kist.re.kr [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of)

    2005-04-01

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn{sup 2+} ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (V{sub M}) of 3.3 Å{sup 3} Da{sup −1} and a solvent content of 62.7%.

  20. DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.

    Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli

    2014-02-15

    A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies. PMID:24035856

  1. Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica

    Storey Kenneth B

    2008-04-01

    Full Text Available Abstract Background The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Results Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0 and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C and by the presence/absence of the natural cryoprotectant (250 mM glucose that

  2. Adenosine-induced activation of esophageal nociceptors.

    Ru, F; Surdenikova, L; Brozmanova, M; Kollarik, M

    2011-03-01

    Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A(1) receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A(1) and/or A(2A) receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A(1) receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A(2A) receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A(1) receptor while the placodes

  3. Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype induced by the adenosine deaminase inhibitor 2'-deoxycoformycin.

    Hershfield, M S; Kurtzberg, J; Harden, E; Moore, J O; Whang-Peng, J; Haynes, B. F.

    1984-01-01

    Selective failure of lymphoid development occurs in genetic deficiency of adenosine deaminase (ADA). We examined the in vivo effects of a potent inhibitor of ADA, 2'-deoxycoformycin, which was used to treat a patient with refractory acute leukemia. Unexpectedly, within 7 days of starting treatment, the leukemic phenotype underwent complete conversion from T lymphoblastic to promyelocytic, with kinetics that suggested a precursor-product relationship between the two cell populations. Pretreatm...

  4. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  5. Escherichia coli ASKA Clone Library Harboring tRNA-Specific Adenosine Deaminase (tadA Reveals Resistance towards Xanthorrhizol

    Yogiara

    2015-09-01

    Full Text Available Xanthorrhizol is a potent antimicrobial compound isolated from the rhizome of Curcuma xanthorrhiza. However, the mechanism of xanthorrhizol action is unknown. To screen for probable target(s, we introduced the ASKA pooled-plasmid library into Escherichia coli W3110 imp4213 and enriched the library for resistant clones with increasing concentrations of xanthorrhizol. After three rounds of enrichment, we found nine genes that increased xanthorrhizol resistance. The resistant clones were able to grow in LB medium containing 256 µg/mL xanthorrhizol, representing a 16-fold increase in the minimum inhibitory concentration. Subsequent DNA sequence analysis revealed that overexpression of tadA, galU, fucU, ydeA, ydaC, soxS, nrdH, yiiD, and mltF genes conferred increased resistance towards xanthorrhizol. Among these nine genes, tadA is the only essential gene. tadA encodes a tRNA-specific adenosine deaminase. Overexpression of E. coli W3110 imp4213 (pCA24N-tadA conferred resistance to xanthorrhizol up to 128 µg/mL. Moreover, overexpression of two tadA mutant enzymes (A143V and F149G led to a twofold increase in the MIC. These results suggest that the targets of xanthorrhizol may include tadA, which has never before been explored as an antibiotic target.

  6. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010

  7. G22A Polymorphism of Adenosine Deaminase and its Association with Biochemical Characteristics of Gestational Diabetes Mellitus in an Iranian Population

    Mohammad Ali Takhshid

    2015-03-01

    Full Text Available Adenosine deaminase (ADA is an important regulator of insulin action. The single nucleotide polymorphism (SNP G22A in the ADA gene decreases enzymatic activity of ADA. The aim of this study was to investigate the relationship between the SNP G22A and blood glycemic control, insulin resistance, and obesity of gestational diabetes mellitus (GDM patients in an Iranian population. SNP G22A was determined in women with GDM (N=70 and healthy pregnant women (control, N=70 using polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma glucose (FPG, hemoglobin A1C (HbA1c, plasma insulin levels and plasma lipids were measured using commercial kits. Homeostasis model of assessment for insulin resistance (HOMA-IR was calculated. The distribution of genotypes and alleles among GDM patients was similar to that of the control group. FPG and HbA1c were significantly higher in GDM patients with GG genotype compared with GDM patients with GA+AA genotype and non-GDM patients. The frequency of GG genotype was significantly higher in obese GDM patients compared to lean GDM patients. The SNP G22A in the ADA gene was not associated with the risk of GDM in our population. GG genotype was associated with poor glycemic control and obesity in GDM patients.

  8. Diagnostic value of serum adenosine deaminase levels in sputum smear negative pulmonary tuberculosis patients in Nepalese population

    Anil Chander; Chandrika Devi Shrestha

    2012-01-01

    Objective: To assess the levels of adenosine deaminase (ADA) in serum in patients with sputum smear negative pulmonary tuberculosis (SNPTB) and to compare it with serum ADA levels in patients with non-tuberculous pulmonary disease - chronic obstructive pulmonary disease (COPD) and with healthy control group and to explore its validity as a diagnostic marker in serum in SNPTB patients.Methods:Three groups of study populations were made. Group I: SNPTB - 142 cases, Group II:non-tubercular pulmonary disease - COPD - 40 cases, Group III: healthy controls - 80 cases. Serum samples were collected and ADA assay was done by the method of Guisti and Galanti. Results: ADA levels (Mean±SD, U/L) in the three groups were as follows: Group I: 42.26±21.22, Group II: 23.31±8.22, Group III: 18.88±6.67. Difference between Group I and Group III was statistically significant (P < 0.0001). The test showed a high specificity 91.25% (95% confidence interval - CI 83.00 - 95.7) and a sensitivity of 83.10% (95% CI 76.08-88.37) in Group I. Positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy in Group I were 94.00%, 69.52%, 9.49, 0.18 and 82.43% respectively.Conclusions: Overall assessment of the use of serum ADA levels as a diagnostic biochemical marker in smear-negative pulmonary tuberculosis patients showed promising results. Studies with a larger population group are required to validate its use as a routine diagnostic test in these cases.

  9. SUB-LETHAL INJECTION OF COBRA VENOM DECREASES ADENOSINE DEAMINASE, HISTAMINE, AND IgE IN ORGANS OF MICE

    B. V. LIPPS

    2002-01-01

    Full Text Available The pharmacological substances adenosine deaminase (ADA, histamine, and IgE are endogenously present in animals. They are implicated in allergy and asthma and detectable in blood serum. This research reports the presence of ADA, histamine, and IgE at varying levels in almost all major organs of mice. This research further reports that intramuscular injection of sub-lethal dose of Naja kaouthia venom disrupted homeostasis and lowered the levels of ADA, histamine, and IgE in the organs of mice. Adult Balb/C male mice were injected with a half lethal dose of the venom. The mice were sacrificed at 2, 8, and 24 hours post-injection and different organs were collected. Organs were homogenized, centrifuged, and the supernatants were assayed for ADA, histamine, and IgE using respective antisera by immunological test enzyme-linked immunosorbent assay (ELISA. Organs from mice injected with PBS served as controls. No major decrease in the levels of ADA, histamine, and IgE was observed after 2 hours of venom injection. However, tremendous decreases in the levels of ADA, histamine, and IgE was observed in organs 24 h post-injection. The highest decrease for ADA was observed in the brain, liver, lung, muscle, and testis; for histamine, in the heart, muscle, lung, and testis; and for IgE, in the bone, heart, lung, muscle, and testis. This is a first-hand investigation showing the effect of envenomation on the pharmacokinetics of ADA, histamine, and IgE in organs.

  10. Acute intermittent porphyria in two patients on anticonvulsant therapy and with normal erythrocyte porphobilinogen deaminase activity.

    Herrick, A. L.; McColl, K E; Moore, M R; Brodie, M J; Adamson, A R; Goldberg, A

    1989-01-01

    1. Acute intermittent porphyria (AIP) is sometimes termed a 'pharmacogenetic' disease. patients with genetic deficiency of the enzyme porphobilinogen deaminase are liable to develop acute attacks of porphyria if exposed to a variety of drugs. 2. Two patients are reported who had no evidence of deficiency of erythrocyte porphobilinogen deaminase yet developed typical attacks of AIP while on anticonvulsant therapy. 3. Normal activity of erythrocyte porphobilinogen deaminase does not completely ...

  11. Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence.

    Frydman, R B; Frydman, B

    1979-06-01

    The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined. PMID:16660874

  12. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  13. EFFICACY OF PLEURAL FLUID ADENOSINE DEAMINASE AND C -REACTIVE PROTEIN LEVELS IN EARLY DIFFERENTIAL DIAGNOSIS OF P LEURAL EFFUSION.

    Aliya

    2013-05-01

    Full Text Available ABSTRACT: - CONTEXT: Pleural effusion occurs secondary to various disea ses. Common causes of exudative effusion are tuberculosis, bacterial pneu monia, and malignancy. Transudative effusion is due to systemic diseases like cardiac failure, cirr hosis of liver. Conventional methods of diagnosis may not be able to establish the cause of pleural e ffusion. Early diagnosis and management reduces the morbidity and mortality. AIM: The objective of the study is to estimate pleural fluid Adenosine Deaminase (ADA and C - reactive protein (CRP leve ls and to evaluate their efficacy in differential diagnosis of transudative and exudative, tuberculou s and non tuberculous and inflammatory and non inflammatory effusions. MATERIAL AND METHODS: Fifty two patients of pleural effusion were investigated and divided into four groups base d on diagnosis. Group I, II, III and IV had 24 cases of tuberculous effusion, 13 cases of transuda tive effusion, 08 cases of malignant effusion and 07 cases of parapneumonic effusion respectively. Pl eural fluid was analyzed for ADA (Guisti and Galanti’s method and CRP (turbidometric immunoassa y. STATISTICAL ANALYSIS: The statistical analysis was done using unpaired student‘t’ test an d p value < 0.05 was considered statistically significant. RESULTS: In the present study pleural fluid ADA revealed hig hly significant increase in tuberculous effusion than non tuberculous effusions (p <0.001 and also when compared with non tuberculous subgroups, transudative effusion (p < 0 .001, malignant effusion (p<0.001, and PPE (p<0.01. ADA levels at a cutoff value of 40U/L, sh owed sensitivity, specificity, positive predictive value and negative predictive value of 91.67%, 89.3 %, 88% & 92.6% respectively in tuberculous effusion. Pleural fluid CRP levels in parapneumonic effusion were significantly higher compared to other types of effusions (p<0.001. Significantly h igher levels of CRP were seen in exudative effusion compared to

  14. Combined detections of interleukin 27, interferon-γ, and adenosine deaminase in pleural effusion for diagnosis of tuberculous pleurisy

    WU Yan-bin; YE Zhi-jian; QIN Sou-ming; WU Cong; CHEN Yi-qiang; SHI Huan-zhong

    2013-01-01

    Background Previous studies reported interleukin-27 (IL-27),interferon-γ (IFN-γ),or adenosine deaminase (ADA) alone plays a helpful role in diagnosing tuberculous pleural effusion (TPE).The present study aims at comparing the diagnostic accuracy of pleural IL-27,IFN-γ,and ADA,and investigate the diagnostic accuracy of the combination of IL-27,IFN-γ,or/and ADA for differentiating TPE from pleural effusions with the other etiologies.Methods The concentrations of IL-27,IFN-γ and ADA were simultaneously determined in pleural fluids and sera from 40 patients with TPE; 26 with malignant pleural effusion,seven with infectious pleural effusion,and eight with transudative pleural effusion by enzyme linked immunosorbent assay and colorimetric method.The corresponding biochemical indexs were also simultaneously determined.Results The concentrations of pleural IL-27 and IFN-γ in the tuberculous group were significantly higher than those in the malignant,infectious,and transudative groups.The concentrations of ADA in TPE were significantly higher than those in MPE or transudative effusions,while much lower than those in infectious effusions.Among these three biomarkers,IL-27 was the most effective for TPE diagnosis,with the cut off value of 900.8 ng/L.IL-27 had a high sensitivity of 95% and specificity of 97.6% for differential diagnosis of TPE from non-TPEs.Combinations of IL-27,IFN-γ and ADA measurements further increased the sensitivity or specificity up to 100%.Conclusions Compared to non-TPEs,IL-27,IFN-γ and ADA all simultaneously increased in TPE; and among these three rapid detection methods,IL-27 appeared to be the best for distinguishing tuberculous from non-TPEs,especially from MPE.Combinations of the three markers (IL-27,IFN-γ and ADA) yielded the highest sensitivity and specificity.These findings suggest that the applications of a new biomarker,IL-27,alone or with IFN-γ and ADA,may contribute to more efficient diagnosis strategies in the management

  15. Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with...

  16. Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes*

    McDougall, William M.; Okany, Chinelo; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function coul...

  17. Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity

    McDougall, William M.; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cy...

  18. Validação laboratorial de um método automatizado de dosagem da atividade de adenosina desaminase em líquido pleural e em líquido cefalorraquidiano Laboratorial validation of an automated assay for the determination of adenosine deaminase activity in pleural fluid and cerebrospinal fluid

    Márcia Cristina Feres

    2008-12-01

    determination of adenosine deaminase (ADA activity in pleural fluid (PF and cerebrospinal fluid (CSF, comparing it with a conventional method (the modified Giusti method. METHODS: In total, 134 samples were selected from among those tested in our laboratory: 94 PF samples and 40 CSF samples. The ADA activity was determined using the two methods. Inter- and intra-assay precision was determined, linear regression analysis was performed, simple concordance tests were conducted, and the means of the differences were calculated. RESULTS: The correlation coefficients for PF and CSF samples were, respectively, 0.96 and 0.95. Inter-assay precision was determined using 21 replicates at 3 different activity levels: low, medium and high. The percentage coefficient of variation (%CV was, respectively, 5.9, 8.1 and 5.8 for PF samples, compared with 21.9, 18.6 and 13.8 for CSF samples. Intra-assay precision in %CV was 1.3 and 11.7, respectively, for PF and CSF samples. The concordance between the methods in PF and CRF samples was, respectively, 96.8% and 100%, considering the reference values for the diagnosis of TB to be 40 U/L (conventional and 30 U/L (automated in PF samples, versus 9 U/L (for both methods in CSF samples. CONCLUSIONS: The results validate the use of the automated method of determining ADA activity in PF and CSF samples as an alternative to the conventional method.

  19. Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes*

    McDougall, William M.; Okany, Chinelo; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription. PMID:21737457

  20. Role of Activation-Induced Cytidine Deaminase in the Development of Oral Squamous Cell Carcinoma

    Yosuke Nakanishi; Satoru Kondo; Naohiro Wakisaka; Akira Tsuji; Kazuhira Endo; Shigeyuki Murono; Makoto Ito; Kouichi Kitamura; Masamichi Muramatsu; Tomokazu Yoshizaki

    2013-01-01

    PURPOSE: In humans, activation-induced cytidine deaminase (AID) expression results due to inflammation and this deaminase activity is also involved in carcinogenesis. The aim of this study is to investigate the correlation between AID expression and the clinical classification of oral cancer tissues. EXPERIMENTAL DESIGN: The current study investigated the correlation between AID expression and the clinical classification of oral cancer tissues from 27 patients who underwent surgical resection...

  1. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    Cátia Vieira; Maria Teresa Magalhães-Cardoso; Fátima Ferreirinha; Isabel Silva; Ana Sofia Dias; Julie Pelletier; Jean Sévigny; Paulo Correia-de-Sá

    2014-01-01

    Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis ...

  2. Biological function of activation-induced cytidine deaminase (AID

    Ritu Kumar

    2014-10-01

    Full Text Available Activation-induced Cytidine Deaminase (AID is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease.

  3. Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity

    McDougall, William M.; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cytoplasmic P-bodies and stress granules. Using a defined in vitro system, we show that RNA alone was sufficient to suppress A3G deaminase activity and did so in an RNA concentration-dependent manner. RNAs of diverse sequences and as short as 25 nucleotides were effective inhibitors. Native PAGE analyses showed that RNA formed ribonucleoprotein complexes with A3G and in so doing prevented ssDNA substrates from binding to A3G. The data provided direct evidence that A3G binding to cellular RNAs constituted a substantial impediment to the enzyme’s ability to interact with ssDNA. PMID:21856286

  4. A combination of the QuantiFERON-TB Gold In-Tube assay and the detection of adenosine deaminase improves the diagnosis of tuberculous pleural effusion.

    Liu, Yuanyuan; Ou, Qinfang; Zheng, Jian; Shen, Lei; Zhang, Bingyan; Weng, Xinhua; Shao, Lingyun; Gao, Yan; Zhang, Wenhong

    2016-01-01

    The differential diagnosis of tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE) remains difficult despite the availability of numerous diagnostic tools. The current study aimed to evaluate the performance of the whole blood QuantiFERON-TB Gold In-Tube (QFT-GIT) assay and conventional laboratory biomarkers in differential diagnosis of TPE and MPE in high tuberculosis prevalence areas. A total of 117 patients with pleural effusions were recruited, including 91 with TPE and 26 with MPE. All of the patients were tested with QFT-GIT, and the conventional biomarkers in both blood and pleural effusion were detected. The level of antigen-stimulated QFT-GIT in the whole blood of TPE patients was significantly higher than that of MPE (2.89 vs 0.33 IU/mL, PGIT for the diagnosis of TPE were 93.0% and 60.0%, respectively. Among the biomarkers in blood and pleural effusion, pleural adenosine deaminase (ADA) was the most prominent biomarker, with a cutoff value of 15.35 IU/L. The sensitivity and specificity for the diagnosis of TPE were 93.4% and 96.2%, respectively. The diagnostic classification tree from the combination of these two biomarkers was 97.8% sensitive and 92.3% specific. Ultimately, the combination of whole blood QFT-GIT with pleural ADA improved both the specificity and positive predictive value to 100%. Thus, QFT-GIT is not superior to pleural ADA in the differential diagnosis of TPE and MPE. Combined whole blood QFT-GIT and pleural ADA detection can improve the diagnosis of TPE. PMID:27485497

  5. 肝病患者血清腺苷脱氨酶检测的临床意义%Study of the serum adenosine deaminase level in patients with hepatitis

    马士恒; 杨彦改; 常亚青; 谢晓华

    2013-01-01

    Objective To study the level of serum adenosine deaminase in patients viral hepatitis. Methods The adenosine deaminase and liver function indexes of the patients were determined by OLYMPUS AU400. Results The rates of increasing serum adenosine deaminase in acute hepatitis,chronic hepatitis, cirrhosis, hepatitis gravis were 55%, 43.8%, 87.9%and 70%, respectively. It was higher in cirrhosis than acute hepatitis, chronic hepatitis(P=0.008, 0.000, respectively). The level of serum adenosine deaminase in cirrhosis patients was higher than that in acute hepatitis patients, chronic hepatitis patients(P=0.003). The serum adenosine deaminase was correlated positively with serum asparate aminotransferase, globulin, the width of portal vein and the thickness of apleen(r=0.214, 0.376, 0.424, 0.306, respectively), it was correlated negatively with serum albumin and cholinesterase (r=-0.405,-0.351, respectively). Conclusion In patients with viral hepatitis, the higher serum adenosine deaminase expressed the decrease of liver synthesization function, it could not reflect the harm of liver cells and cholestasis perfectly.%  目的研究病毒性肝炎患者血清腺苷脱氨酶(ADA)水平.方法应用全自动生化分析仪检测各型病毒性肝炎患者血清ADA及各项肝功能指标.结果急性肝炎、慢性肝炎、肝炎后肝硬化、重型肝炎患者血清ADA升高比例分别为55%、43.8%、87.9%及70%,肝炎后肝硬化组显著高于急性肝炎组、慢性肝炎组(P分别为0.008、0.000).肝炎后肝硬化组ADA血清水平亦显著高于急性肝炎组、慢性肝炎组(P均为0.003).病毒性肝炎患者血清ADA与门冬氨酸转氨酶、球蛋白、门静脉宽度、脾脏厚度呈显著正相关(r分别为0.214、0.376、0.424、0.306),与白蛋白、胆碱脂酶呈显著负相关(r分别为-0.405、-0.351).结论在病毒性肝炎患者中,血清ADA升高主要反映肝脏储备合成功能逐渐降低,而不能很好反映肝细胞受损及胆汁淤积.

  6. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  7. A coming-of-age story: activation-induced cytidine deaminase turns 10

    Delker, Rebecca K; Fugmann, Sebastian D.; Papavasiliou, F. Nina

    2009-01-01

    The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implica...

  8. Value of combined detection of interferon-γ, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion

    Objective: To explore the value of interferon II, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion. Methods: 122 cases with tuberculous pleurisy, 56 cases of malignant pleural effusion, 48 cases of tuberculous pleural effusion, 18 cases of inflammatory and other pleural fluid were studied. The serum and pleural fluid levels of IFN-γ, VEGF-C, CRP and ADA serum in those patients were detected. Results: The IFN-γ, CRP and ADA levels in tuberculous pleural effusion were higher than in malignant pleural effusion(P<0.01). According to the receiver operator characteristic (ROC) curve, when 100 ng/L was regarded as critical value of IFN-γ, the sensitivity and specificity of IFN-γ in diagnosing tuberculous pleural effusion were 83.1% and 92.3% respectively. When 45 U/L ADA was regarded as critical value of ADA, the sensitivity and specificity of ADA in diagnosing tuberculous pleural effusion were 85.6% and 96.3% respectively. When 110 mg/L was regarded as critical value of CRP, the sensitivity and specificity of CRP were 79.1% and 84.2% respectively. When combine detection of three markers, the diagnosis sensitivity and specificity were 87.8% and 86.0% respectively. The VEGF-C concentration in malignant pleural effusion was higher than that in tuberculous pleural effusion and inflammatory and other pleural effusion (P<0.01). When the ratio of VEGF-C to ADA≥8, the sensitivity and specificity in diagnosis of malignant pleural effusion were 86.3% and 82.6% respectively, and the ration VEGF-C to ADA≤3, the sensitivity and specificity in diagnosis of tuberculous pleural effusion were 85.1% and 87.1% respectively. Conclusion: The combined detection of IFN-γ, CRP and ADA could improve sensitivity and specificity in diagnosing tuberculous pleurisy. The concentration ratios of VEGF-C to ADA have clinical value in differential diagnosis of pleural effusions. (authors)

  9. In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

    Newman, E. B.; D. Dumont; Walker, C.

    1985-01-01

    Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene produ...

  10. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P.; Stone, T W

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked ...

  11. Activity and immunohistochemical localization of porphobilinogen deaminase in rat tissues

    Jørgensen, P E; Erlandsen, E J; Poulsen, Steen Seier;

    2000-01-01

    the activity and the immunohistochemical localization of PBGD in the following tissues of wistar female rats: brain, heart, submandibular gland, liver, kidney, pancreas, ovary, stomach, duodenum, jejunum, ileum, colon and musculature. The PBGD activity varied considerably among the tissues. It was highest...... in the intestine. Especially, the immunohistochemical reaction in nerve cells is notable considering the nervous dysfunction in AIP....

  12. A novel L-serine deaminase activity in Escherichia coli K-12.

    Su, H. (Hsin-Ning); Newman, E B

    1991-01-01

    We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in m...

  13. Activation-induced cytidine deaminase acts on double-strand breaks in vitro.

    Shen, Hong Ming

    2007-02-01

    Activation-induced cytidine deaminase (AID) is likely responsible for DNA cytidine deamination, although it may also act as an RNA deaminase. It functions on single-stranded DNA, the non-template strand in double-stranded DNA during transcription, or both strands in supercoiled DNA. To ask whether AID is able to deaminate cytidine at DNA breaks, plasmids, containing a SnaBI site (TAC downward arrowGTA) that forms blunt ends after digestion with SnaBI, were generated. If AID deaminates cytidine at the upstream blunt end, the ATG start codon in either of two drug resistance genes will be regenerated after ligation and replication in UDG-null E. coli cells. This study shows that AID targets cytidine at the break. The extent of deamination activity beyond the break is correlated with the base composition in the break region. If the break region is A, T-rich, C > T transitions are extensive. However, when the break region is not A, T-rich, mutations are mainly restricted to the break, similar to findings in vivo. The results indicate that AID has activity on double strand breaks (DSBs). Based on previous and current findings, a somatic hypermutation (SHM) model is proposed, in which collision between the transcription apparatus and the replication fork generates DSBs. After AID acts on break ends, the error-prone DNA repair machinery fixes and creates mutations. PMID:16697045

  14. Dosagem da atividade da adenosina deaminase no líquido pleural para o diagnóstico da tuberculose pleural Pleural fluid adenosine deaminase detection for the diagnosis of pleural tuberculosis

    Morrys Casagrande Kaisemann; Afrânio Lineu Kritski; Maria de Fátima C Pereira; Anete Trajman

    2004-01-01

    INTRODUÇÃO: O diagnóstico da tuberculose pleural permanece um desafio, pois a sensibilidade dos testes tradicionais é baixa. O exame histopatológico da pleura é o método mais preciso, com até 80% de sensibilidade. A dosagem da adenosina deaminase foi introduzida mais recentemente, mas sua utilidade no diagnóstico da tuberculose pleural no Brasil não foi suficientemente esclarecida. OBJETIVO: Verificar a sensibilidade e a especificidade de um método experimental de dosagem da atividade da aden...

  15. Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis

    Stav Sapoznik

    2016-02-01

    Full Text Available In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection.

  16. Acquisition of Genetic Aberrations by Activation-Induced Cytidine Deaminase (AID) during Inflammation-Associated Carcinogenesis

    Genetic abnormalities such as nucleotide alterations and chromosomal disorders that accumulate in various tumor-related genes have an important role in cancer development. The precise mechanism of the acquisition of genetic aberrations, however, remains unclear. Activation-induced cytidine deaminase (AID), a nucleotide editing enzyme, is essential for the diversification of antibody production. AID is expressed only in activated B lymphocytes under physiologic conditions and induces somatic hypermutation and class switch recombination in immunoglobulin genes. Inflammation leads to aberrant AID expression in various gastrointestinal organs and increased AID expression contributes to cancer development by inducing genetic alterations in epithelial cells. Studies of how AID induces genetic disorders are expected to elucidate the mechanism of inflammation-associated carcinogenesis

  17. A coming-of-age story: activation-induced cytidine deaminase turns 10.

    Delker, Rebecca K; Fugmann, Sebastian D; Papavasiliou, F Nina

    2009-11-01

    The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implications of AID function on human physiology. We also take time to point out important ongoing controversies and outstanding questions in the field and highlight key experiments and techniques that hold the potential to elucidate the remaining mysteries surrounding this vital protein. PMID:19841648

  18. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  19. Avaliação da adenosina desaminase no diagnóstico da tuberculose pleural: uma metanálise brasileira Evaluation of adenosine deaminase in the diagnosis of pleural tuberculosis: a Brazilian meta-analysis

    Patrizio Morisson

    2008-04-01

    Full Text Available OBJETIVO: Avaliar trabalhos brasileiros resumindo a acurácia da adenosina desaminase no diagnóstico da tuberculose pleural, com o intuito de contribuir para a concretização do exame como rotina na investigação dos derrames pleurais. MÉTODOS: Depois de realizada uma busca por trabalhos brasileiros referentes à dosagem da adenosina desaminase no líquido pleural, estes foram avaliados e incluídos no estudo. A análise dos dados foi feita por meio da curva summary receiver operating characteristic (SROC que possibilitou a reunião dos estudos quanto a acurácia para o diagnóstico. Com os valores globais de sensibilidade e especificidade foi aplicado o teorema de Bayes para calcular as probabilidades pós-teste em diferentes prevalências da doença. RESULTADOS: Entre 1987 e 2005 foram encontrados 25 estudos contendo informações suficientes que poderiam ser utilizadas na metanálise. Após avaliação, foram incluídos nove estudos, totalizando 1.674 pacientes. De acordo com a curva SROC, foi encontrada uma sensibilidade de 91,8% (IC95%: 89,8-93,6% e uma especificidade de 88,4% (IC95%: 86,0-90,5%, com uma área abaixo da curva de 0,969. O odds ratio global foi de 112,0 (IC95%: 51,6-243,2. Considerando uma prevalência da tuberculose pleural de 50% (considerada neutra, a probabilidade do diagnóstico pós-teste positivo é de 88,7% e sua exclusão após resultado negativo de 91,5%. CONCLUSÕES: Apesar das diferenças encontradas entre os estudos, é possível concluir que a adenosina desaminase possui alta acurácia no diagnóstico da tuberculose pleural, devendo ser utilizada de rotina em sua investigação.OBJECTIVE: To evaluate Brazilian studies by summarizing the accuracy of adenosine deaminase in the diagnosis of pleural tuberculosis, with the objective of lending support to the movement to make the test part of the routine investigation of pleural effusions. METHODS: A search for Brazilian studies related to the determination of

  20. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( Panimals.

  1. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  2. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation

  3. Significance of determination of serum cytidine deaminase (CD) levels for diagnosis of active rheumatoid arthritis (RA)

    Objective: To determine the clinical value of measurement of serum cytidine deaminase (CD) levels in patients with active rheumatoid arthritis (RA). Methods: Serum levels of CD were detected with spectrophotometry, in 33 patients with active RA and 60 controls. The erythrocyte sedimentation rate (ESR) and CRP content were also determined in both groups. Results: The ser- um CD contents in patients with active RA(14.80 ± 2.11U/ml) were significantly higher than those in controls(4.86±1.86 U/ml,P<0.01). The CRP contents (51.46 ± 20.43mg/L) and ESR readings(85.03 ± 27.6mm/h) in the patients were also significantly higher than those in the controls(3.40 ± 2.21mg/L and 13.04 ± 4.89mm/h respectively, all P<0.01). In the patients, the serum CD contents were linearly positively correlated with the ESR contents and CRP readings (r=0.6324 and 0.8013 respectively, P <0.01). Conclusion: Serum CD is an early biochemical marker for diagnosis of active rheumatoid arthritis and is also of prognostic value. (authors)

  4. Characterization and optimization of 1-Aminocyclopropane-1-Carboxylate Deaminase (ACCD activity in different rhizospheric PGPR along with Microbacterium sp. strain ECI-12A

    Ashok Kumar

    2013-03-01

    Full Text Available A total of nine strains of plant growth promoting rhizobacteria were analyzed for ACC deaminase activity, where highest ACC deaminase activity was found in Klebsiella sp strain ECI-10A (539.1 nmol α-keto butyrate/ mg protein/ h and lowest in Microbacterium sp strain ECI-12A (122.0 nmol α-keto butyrate/ mg protein/ h. Although Microbacterium sp strain ECI-12A showed lowest level of ACC deaminase activity, but, the species of Microbacterium isolated from rhizosphere is the first report. Microbacterium sp strain ECI-12A was also analyzed under varying conditions of time, amount of 1-Aminocyclopropane-1- carboxylate (ACC, and temperature for optimization of the ACC deaminase activity. The optimum activity was recorded with the supplementation of 5mM ACC at 30oC temperature after 24h of culture growth. All the nine strains showed acdS gene in the PCR amplification of that gene. No any rhizospheric Microbacterium species showing ACC deaminase activity have been reported earlier, therefore, we report here ACC deaminase activity in Microbacterium sp ECI-12A isolated from rice rhizosphere is a novel finding.

  5. Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase

    Hu, Wenjun; Begum, Nasim A.; Mondal, Samiran; Stanlie, Andre; Honjo, Tasuku

    2015-01-01

    The B-cell–specific antibody gene-diversifying enzyme activation-induced cytidine deaminase (AID) shows high homology with apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC1), which edits apolipoprotein B-100 mRNA in the presence of cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Here we show that the DNA cleavage and recombination functions of AID depend critically on its RNA-dependent interaction with distinct heterogeneous nuclear ribonu...

  6. Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification.

    Pham, Phuong; Afif, Samir A; Shimoda, Mayuko; Maeda, Kazuhiko; Sakaguchi, Nobuo; Pedersen, Lars C; Goodman, Myron F

    2016-07-01

    Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating C→U during transcription of Ig-variable (V) and Ig-switch (S) region DNA, which is essential to produce high-affinity antibodies. Here we report the crystal structure of a soluble human AID variant at 2.8Å resolution that favors targeting WRC motifs (W=A/T, R=A/G) in vitro, and executes Ig V SHM in Ramos B-cells. A specificity loop extending away from the active site to accommodate two purine bases next to C, differs significantly in sequence, length, and conformation from APOBEC proteins Apo3A and Apo3G, which strongly favor pyrimidines at -1 and -2 positions. Individual amino acid contributions to specificity and processivity were measured in relation to a proposed ssDNA binding cleft. This study provides a structural basis for residue contributions to DNA scanning properties unique to AID, and for disease mutations in human HIGM-2 syndrome. PMID:27258794

  7. ACC (1-Aminocyclopropane-1-Carboxylate) Deaminase Activity, a Widespread Trait in Burkholderia Species, and Its Growth-Promoting Effect on Tomato Plants▿

    Onofre-Lemus, Janette; Hernández-Lucas, Ismael; Girard, Lourdes; Caballero-Mellado, Jesús

    2009-01-01

    The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are...

  8. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  9. Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease.

    Koji Hase

    Full Text Available Activation-induced cytidine deaminase (AID expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM and class switch recombination (CSR. Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2 associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

  10. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  11. Role of adenosine in the sympathetic activation produced by isometric exercise in humans.

    Costa, F.; Biaggioni, I

    1994-01-01

    Isometric exercise increases sympathetic nerve activity and blood pressure. This exercise pressor reflex is partly mediated by metabolic products activating muscle afferents (metaboreceptors). Whereas adenosine is a known inhibitory neuromodulator, there is increasing evidence that it activates afferent nerves. We, therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressor reflex in healthy volunteers. Intraarterial administration of...

  12. Selective detection of adenosine A1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [35S]guanosine 5minutes or feet-(γ-thio)triphosphate autoradiography

    [35S]Guanosine 5minutes or feet-(γ-thio)triphosphate autoradiography is a novel technique to detect receptor-dependent activation of G-proteins in brain tissue sections. While an increasing number of reports using this approach are beginning to appear, little effort has been directed to the identification of factors responsible for the heterogeneously distributed [35S]guanosine 5minutes or feet-(γ-thio)triphosphate signal in basal conditions. The present study demonstrates that endogenously formed adenosine generates a widespread and prominent adenosine A1 receptor-dependent signal in basal conditions using this technique. Treatment of rat brain tissue sections with the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine dose-dependently (ec5035S]guanosine 5minutes or feet-(γ-thio)triphosphate binding in a region-specific manner, an effect fully mimicked by the adenosine-depleting enzyme adenosine deaminase, and less so by the A1 antagonist cirsimarin and by caffeine. That adenosine was continuously formed during the incubation is supported by the constant requirements of adenosine deaminase in order to suppress basal radioligand binding and further by the fact that low micromolar concentrations of adenine nucleotides evoked only adenosine-mimicking and fully 8-cyclopentyl-1,3-dipropylxanthine-sensitive binding responses. In the presence of adenosine deaminase, all responses to adenine nucleotides were abolished, indicating that prior conversion to adenosine was required. Upon stimulation, this technique selectively detected A1 receptor-activated G-proteins, as the non-selective agonists adenosine and 2-chloroadenosine and the A1-selective agonist N6-p-sulfophenyladenosine all evoked only 8-cyclopentyl-1,3-dipropylxanthine-sensitive responses in identical gray matter areas, and also in several white matter areas such as the corpus callosum, anterior commissure, optic tract and cerebellar white matter. Dose-response studies revealed region

  13. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

  14. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    Miguel A Lanaspa

    Full Text Available Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2 (summer and activation of AMP-activated protein kinase (AMPK (winter. Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2, as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation. Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC and decreased enoyl CoA hydratase (ECH1 and carnitine palmitoyltransferase 1A (CPT1A, rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

  15. Adenosine, ketogenic diet and epilepsy: the emerging therapeutic relationship between metabolism and brain activity.

    Masino, S A; Kawamura, M; Wasser, C D; Wasser, C A; Pomeroy, L T; Ruskin, D N

    2009-09-01

    For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a "retaliatory metabolite." As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor-based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed. PMID:20190967

  16. Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates.

    Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S; Schatz, David G

    2007-08-31

    The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. PMID:17613522

  17. Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

    Aliagas, Elisabet; Villar-Menéndez, Izaskun; Sévigny, Jean; Roca, Mercedes; Romeu, Miriam; Ferrer, Isidre; Martín-Satué, Mireia; Barrachina, Marta

    2013-01-01

    Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5′-nucleotidase, and alkaline phosphatase) in the postmortem putame...

  18. Suppression of adenosine-activated chloride transport by ethanol in airway epithelia.

    Sammeta V Raju

    Full Text Available Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B adenosine receptor (A(2BAR, largely abolished the adenosine-stimulated chloride transport, suggesting that A(2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

  19. T-Independent Activation-Induced Cytidine Deaminase Expression, Class-Switch Recombination, and Antibody Production by Immature/Transitional 1 B Cells1

    Ueda, Yoshihiro; Liao, Dongmei; Yang, Kaiyong; Patel, Anjali; Kelsoe, Garnett

    2007-01-01

    Inflammation elicits a splenic lymphopoiesis of unknown physiologic significance but one that juxtaposes developing B cells and exogenous Ag. We show that immature and transitional 1 (immature/T1) B cells constitutively express activation-induced cytidine deaminase and B lymphocyte-induced maturation protein 1 in amounts that support accelerated plasmacytic differentiation and limited class-switch recombination. In vivo, activation of immature/T1 B cells by TLR ligands or bacterial vaccine ra...

  20. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  1. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients. PMID:26521170

  2. Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death

    Zuberová, M.; Fencková, M.; Šimek, Petr; Janečková, L.; Doležal, T.

    2010-01-01

    Roč. 3, 11/12 (2010), s. 773-784. ISSN 1754-8403 R&D Projects: GA ČR(CZ) GD204/09/H058 Grant ostatní: AV ČR(CZ) KJB501410602; GA ČR(CZ) GA204/09/1463 Institutional research plan: CEZ:AV0Z50070508 Keywords : central nervous system * glycogen phosphorylase * immune response Subject RIV: CE - Biochemistry Impact factor: 4.584, year: 2010

  3. Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

    Marquez-Rios, E; Pacheco-Aguilar, R; Castillo-Yañez, F J; Figueroa-Soto, C G; Ezquerra-Brauer, J M; Gollas-Galvan, T

    2008-09-01

    Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms. PMID:26050167

  4. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  5. Isotype-switched follicular lymphoma displays dissociation between activation-induced cytidine deaminase expression and somatic hypermutation.

    Scherer, Florian; Navarrete, Marcelo A; Bertinetti-Lapatki, Cristina; Boehm, Joachim; Schmitt-Graeff, Annette; Veelken, Hendrik

    2016-01-01

    In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease. PMID:25860234

  6. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types

  7. Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo.

    Isora V Sernández

    Full Text Available The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR and Somatic Hypermutation (SHM. The enzyme Activation Induced Cytidine Deaminase (AID initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID(+/-. AID(+/- mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID(+/- cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID(+/- mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID(+/- mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity.

  8. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  9. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

    Simon Gemble

    2015-07-01

    Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

  10. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

    Gnad, Thorsten; Scheibler, Saskia; von Kügelgen, Ivar;

    2014-01-01

    Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT...... that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies....

  11. Adenosine induces vasoconstriction through Gi-dependent activation of phospholipase C in isolated perfused afferent arterioles of mice

    Hansen, Pernille B; Castrop, Hayo; Briggs, Josie; Schnermann, Jurgen

    2003-01-01

    Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear. In......-induced vasoconstriction was stable for up to 30 min and was most pronounced in the most distal part of the afferent arterioles. Adenosine did not cause vasoconstriction in arterioles from A1AR-/- mice. Pretreatment with pertussis toxin (PTX) (400 ng/ml) for 2 h blocked the vasoconstricting action of adenosine or N(6......) blocked the constriction responses to both adenosine and angiotensin II. In contrast, the adenylate cyclase inhibitor SQ22536 (10 micro M) and the protein kinase A antagonist KT5720 (0.1 and 1 micro M) did not induce significant vasoconstriction of afferent arterioles. It is concluded that the...

  12. Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

    Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-adenosyl-methionine and S-adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM was shown to induce the membrane phospholipid methylation as assessed by the 3Hmethyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation

  13. Effect of repeated pesticide applications on soil properties in cotton fields: II. Insecticide residues and impact on dehydrogenase and arginine deaminase activities

    Insecticides were applied sequentially at recommended dosages post crop emergence in cotton fields and soil was sampled at regular intervals after each treatment. Soil was analysed for insecticide residues and activity of the enzymes dehydrogenase and arginine deaminase. Insecticide residues detected in the soil were in small quantities and they did not persist for long. Only endosulfan leached below 15 cm. Insecticides had only temporary effects on enzyme activities which disappeared either before the next insecticide treatment or by the end of the experimental period. (author)

  14. Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the "adenosine hypothesis".

    Aliagas, Elisabet; Villar-Menéndez, Izaskun; Sévigny, Jean; Roca, Mercedes; Romeu, Miriam; Ferrer, Isidre; Martín-Satué, Mireia; Barrachina, Marta

    2013-12-01

    Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5'-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5'-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness. PMID:23771238

  15. Respiratory activity in medulla oblongata and its modulation by adenosine and opioids

    Herlenius, Eric

    1998-01-01

    From the moment of birth the complex neuronal networks generating breathing has to function continuously and adapt to the new postnatal environmental demands. This thesis aims at studying the perinatal development of respiratory control and its modulation by adenosine and opioids. Respiratory activity was studied in vitro using brainstem spinal cord preparations and in vivo with a barometric plethysmograph. In vitro whole-cell patch clamp recordings of respiratory related ne...

  16. Biosynthesis of bacterial glycogen: activator specificity of the adenosine diphosphate glucose pyrophosphorylases from the genus Rhodospirillum.

    Preiss, J; Greenberg, E

    1981-01-01

    The adenosine diphosphate (ADP) glucose pyrophosphorylases from Rhodospirillum fulvum, Rhodospirillum molischianum, and Rhodospirillum tenue were partially purified, and their kinetic properties were studied. The enzyme from the three organisms was found to be activated by pyruvate and thus was similar to the Rhodospirillum rubrum enzyme that had been previously studied (C. E. Furlong, and J. Preiss, J. Biol. Chem. 244:2539-2548, 1979). The enzymes from R. fulvum, R. molischianum, and R. tenu...

  17. Repression of human activation induced cytidine deaminase by miR-93 and miR-155

    Activation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis. The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells. We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA. Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent

  18. Repression of human activation induced cytidine deaminase by miR-93 and miR-155

    Holton Nathaniel W

    2011-08-01

    Full Text Available Abstract Background Activation Induced cytidine Deaminase (AID targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis. Methods The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells. Results We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA. Conclusions Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located

  19. Modulation of bladder function by luminal adenosine turnover and A1 receptor activation

    Prakasam, H. Sandeep; Herrington, Heather; Roppolo, James R.; Jackson, Edwin K.; Apodaca, Gerard

    2012-01-01

    The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A1, A2A, A2B, and A3), and is a site of adenosine production. Although adenosine has a well-described protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed ...

  20. Activation of adenosine receptors and inhibition of cyclooxygenases: two recent pharmacological approaches to modulation of radiation suppressed hematopoiesis

    Searching for drugs conforming to requirements for protection and/or treatment of radiation-induced damage belongs to the most important tasks of current radiobiology. In the Laboratory of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic, two original approaches for stimulation of radiation-suppressed hematopoiesis have been tested in recent years, namely activation of adenosine receptors and inhibition of cyclooxygenases. Non-selective activation of adenosine receptors, induced by combined administration of dipyridamole, a drug preventing adenosine uptake and supporting thus its extracellular receptor-mediated action, and adenosine monophosphate, an adenosine prodrug, has been found to stimulate hematopoiesis when the drugs were given either pre- or post-irradiation. When synthetic adenosine receptor agonists selective for individual adenosine receptor subtypes were tested, stimulatory effects in myelosuppressed mice have been found after administration of IB-MECA, a selective adenosine A3 receptor agonist. Non-selective cyclooxygenase inhibitors, inhibiting both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), indomethacin, diclofenac, or flurbiprofen, have been observed to act positively on radiation-perturbed hematopoiesis in sublethally irradiated mice. However, their undesirable gastrointestinal side effects have been found to negatively influence survival of lethally irradiated animals. Recently tested selective COX-2 inhibitor meloxicam, preserving protective action of COX-1-synthesized prostaglandins in the gastrointestinal tissues, has been observed to retain the hematopoiesis-stimulating effects of non-selective cyclooxygenase inhibitors and to improve the survival of animals exposed to lethal radiation doses. These findings bear evidence for the possibility to use selective adenosine A3 receptor agonists and selective COX-2 inhibitors in human practice for treatment of

  1. Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

    Jerome Mauris

    Full Text Available BACKGROUND: Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity. CONCLUSIONS: ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

  2. Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity

    Jerome Mauris; Thomas C Evans

    2009-01-01

    BACKGROUND: Human PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif pre...

  3. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei(Helmholtz-Institut für Strahlen- und Kernphysik, Bethe Center for Theoretical Physics, Universität Bonn, Bonn, D-53115, Germany); Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K.; Blackburn, Michael R.; Kellems, Rodney E.; Xia, Yang

    2014-01-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ...

  4. AID up-mutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity

    Wang, Meng; Yang, Zizhen; Rada, Cristina; Neuberger, Michael S.

    2016-01-01

    DNA deaminases underpin pathways in antibody diversification (AID) and anti-viral immunity (APOBEC3s). Here we show how a high-throughput bacterial papillation assay can be used to screen for AID mutants with increased catalytic activity. The upmutations focus on a small number of residues, some highlighting regions likely implicated in AID’s substrate interaction. Notably, many of the upmutations bring the sequence of AID closer to that of APOBEC3s. AID upmutants can yield increased antibody diversification, raising the possibility that modification of AID’s specific activity might be used to regulate antibody diversification in vivo. However, upmutation of AID also led to increased frequency of chromosomal translocations suggesting that AID’s specific activity may have been limited by the risk of genomic instability. PMID:19543289

  5. Staurosporine-induced apoptosis in astrocytes is prevented by A1 adenosine receptor activation.

    D'Alimonte, Iolanda; Ballerini, Patrizia; Nargi, Eleonora; Buccella, Silvana; Giuliani, Patricia; Di Iorio, Patrizia; Caciagli, Francesco; Ciccarelli, Renata

    2007-05-11

    Astrocyte apoptosis occurs in acute and chronic pathological processes at the central nervous system and the prevention of astrocyte death may represent an efficacious intervention in protecting neurons against degeneration. Our research shows that rat astrocyte exposure to 100 nM staurosporine for 3h caused apoptotic death accompanied by caspase-3, p38 mitogen-ed protein kinase (MAPK) and glycogen synthase kinase-3beta (GSK3beta) activation. N(6)-chlorocyclopentyladenosine (CCPA, 2.5-75 nM), a selective agonist of A(1) adenosine receptors, added to the cultures 1h prior to staurosporine, induced a dose-dependent anti-apoptotic effect, which was inhibited by the A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. CCPA also caused a dose- and time-dependent phosphorylation/activation of Akt, a downstream effector of cell survival promoting phosphatidylinositol 3-kinase (PI3K) pathway, which in turn led to inhibition of staurosporine-induced GSK3beta and p38 MAPK activity. Accordingly, the anti-apoptotic effect of CCPA was abolished by culture pre-treatment with LY294002, a selective PI3K inhibitor, pointing out the prevailing role played by PI3K pathway in the protective effect exerted by A(1) receptor activation. Since an abnormal p38 and GSK3beta activity is implicated in acute (stroke) and chronic (Alzheimer's disease) neurodegenerative diseases, the results of the present study provide a hint to better understand adenosine relevance in these disorders. PMID:17400382

  6. Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.

    Shirali, Saeid; Aghaei, Mahmoud; Shabani, Mahdi; Fathi, Mojtaba; Sohrabi, Majid; Moeinifard, Marzieh

    2013-04-01

    Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer. PMID:23345014

  7. Activation of NTS A1 adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex

    Ichinose, Tomoko K.; Minic, Zeljka; Li, Cailian; O'Leary, Donal S.; Scislo, Tadeusz J.

    2012-01-01

    Previously we have shown that adenosine operating via the A1 receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it i...

  8. Endogenous activation of adenosine A1 receptors promotes post-ischemic electrocortical burst suppression

    Ilie, A; Ciocan, D; Constantinescu, A O; Zagrean, A-M; Nita, D A; Zagrean, L; Moldovan, M

    2009-01-01

    -vessel occlusion" model under chloral hydrate anesthesia. Quantification of BS recovery was carried out using BS ratio. During GCI full electrocortical suppression was attained (BS ratio reached 100%). During the following reperfusion the BS ratio returned to 0. The time course of the decay was exponential after 1...... and 5-min GCI and bi-exponential after 10-min GCI. The BS recovery was progressively delayed with the duration of ischemia. Administration of the A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1.25 mg/kg i.p.) accelerated the post-ischemic BS recovery for all GCI durations. Following the 10......-min GCI the effect of DPCPX was only apparent on the initial fast decay of the BS ratio. These data suggest that endogenous adenosine release promotes BS patterns during reperfusion following transient cerebral ischemia. Furthermore, the endogenous A1R activation may be the primary underlying cause of...

  9. Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor.

    Kobayashi, S; Conforti, L; Pun, R Y; Millhorn, D E

    1998-04-01

    1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in

  10. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL.

    Eydmann, T; Söderbäck, E; Jones, T; Hill, S; Austin, S; Dixon, R

    1995-03-01

    The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch. PMID:7868590

  11. Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

    Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production. The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers

  12. Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

    Kim Samyong

    2005-05-01

    Full Text Available Abstract Background Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12, a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD and 5-fluorocytosine (5-FC has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca tumors. Methods Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12 or cytosine deaminase (Ad.CD. The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK cell activity and IFN-γ production. Results The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control. The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. Conclusion The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by

  13. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  14. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

    de Mendonça, Alexandre; Ribeiro, J. A.

    1997-01-01

    Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of ...

  15. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  16. Activation-induced cytidine deaminase expression in CD4+ T cells is associated with a unique IL-10-producing subset that increases with age.

    Hongyan Qin

    Full Text Available Activation-induced cytidine deaminase (AID, produced by the Aicda gene, is essential for the immunoglobulin gene (Ig alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4(+ T cells that had expressed Aicda (exAID cells as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4(+ and B220(+ cell populations. ExAID B cells remained IgM(+, suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis.

  17. Expression of activation-induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti-pneumococcal B1a cells.

    Yamamoto, Natsuo; Kerfoot, Steven M; Hutchinson, Andrew T; Dela Cruz, Charles S; Nakazawa, Naomi; Szczepanik, Marian; Majewska-Szczepanik, Monika; Nazimek, Katarzyna; Ohana, Noboru; Bryniarski, Krzysztof; Mori, Tsutomu; Muramatsu, Masamichi; Kanemitsu, Keiji; Askenase, Philip W

    2016-01-01

    We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset. PMID:26456931

  18. Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings concomitantly with activation-induced cytidine deaminase expression and differentiation into plasma cells.

    Kobayashi, Shinji; Haruo, Nagumo; Sugane, Kazuo; Ochs, Hans D; Agematsu, Kazunaga

    2009-01-01

    Interleukin (IL)-21 downregulates immunoglobulin E (IgE) production in murine systems by inhibiting germline epsilon transcription in IL-4-stimulated B cells. We here sought to clarify the function of IL-21 in human B-cell IgE synthesis. IL-21 dramatically enhanced IgE production by human mononuclear cells, or purified total, naive, or memory B cells in the presence of IL-4 plus anti-CD40 mAb cross-linked with CD32-transfectants, and the production was strengthened with further addition of IL-10. It was concomitant to the enhancement of activation-induced cytidine deaminase (AID) mRNA expression, but no increase of germline epsilon transcription. We also observed that IL-21 promoted B-cell differentiation into plasma cells with increase of B-lymphocyte-induced maturation protein-1 (Blimp-1), but not X-box binding protein 1 (XBP-1), which was further accentuated by co-stimulation with IL-4 plus CD40 signaling. Thus, IL-21 is a strong inducer of IgE production in human beings concomitantly with AID expression and the differentiation into plasma cells. Our data suggest that IL-21 plays an important role in occurrence and the treatment of allergic disorders. PMID:19026702

  19. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  20. Adenosine can thwart antitumor immune responses elicited by radiotherapy. Therapeutic strategies alleviating protumor ADO activities

    By studying the bioenergetic status we could show that the development of tumor hypoxia is accompanied, apart from myriad other biologically relevant effects, by a substantial accumulation of adenosine (ADO). ADO has been shown to act as a strong immunosuppressive agent in tumors by modulating the innate and adaptive immune system. In contrast to ADO, standard radiotherapy (RT) can either stimulate or abrogate antitumor immune responses. Herein, we present ADO-mediated mechanisms that may thwart antitumor immune responses elicited by RT. An overview of the generation, accumulation, and ADO-related multifaceted inhibition of immune functions, contrasted with the antitumor immune effects of RT, is provided. Upon hypoxic stress, cancer cells release ATP into the extracellular space where nucleotides are converted into ADO by hypoxia-sensitive, membrane-bound ectoenzymes (CD39/CD73). ADO actions are mediated upon binding to surface receptors, mainly A2A receptors on tumor and immune cells. Receptor activation leads to a broad spectrum of strong immunosuppressive properties facilitating tumor escape from immune control. Mechanisms include (1) impaired activity of CD4 + T and CD8 + T, NK cells and dendritic cells (DC), decreased production of immuno-stimulatory lymphokines, and (2) activation of Treg cells, expansion of MDSCs, promotion of M2 macrophages, and increased activity of major immunosuppressive cytokines. In addition, ADO can directly stimulate tumor proliferation and angiogenesis. ADO mechanisms described can thwart antitumor immune responses elicited by RT. Therapeutic strategies alleviating tumor-promoting activities of ADO include respiratory hyperoxia or mild hyperthermia, inhibition of CD39/CD73 ectoenzymes or blockade of A2A receptors, and inhibition of ATP-release channels or ADO transporters. (orig.)

  1. Increased orbitofrontal brain activation after administration of a selective adenosine A2A antagonist in cocaine dependent subjects

    F. Gerard eMoeller

    2012-05-01

    Full Text Available Background: Positron Emission Tomography imaging studies provide evidence of reduced dopamine function in cocaine dependent subjects in the striatum, which is correlated with prefrontal cortical glucose metabolism, particularly in the orbitofrontal cortex. However, whether enhancement of dopamine in the striatum in cocaine dependent subjects would be associated with changes in prefrontal cortical brain activation is unknown. One novel class of medications that enhance dopamine function via heteromer formation with dopamine receptors in the striatum is the selective adenosine A2A receptor antagonists. This study sought to determine the effects administration of the selective adenosine A2A receptor antagonist SYN115 on brain function in cocaine dependent subjects. Methodology/Principle Findings: Twelve cocaine dependent subjects underwent two fMRI scans (one after a dose of placebo and one after a dose of 100 mg of SYN115 while performing a working memory task with 3 levels of difficulty (3, 5, and 7 digits. fMRI results showed that for 7-digit working memory activation there was significantly greater activation from SYN115 compared to placebo in portions of left (L lateral orbitofrontal cortex, L insula, and L superior and middle temporal pole. Conclusion/Significance: These findings are consistent with enhanced dopamine function in the striatum in cocaine dependent subjects via blockade of adenosine A2A receptors producing increased brain activation in the orbitofrontal cortex and other cortical regions. This suggests that at least some of the changes in brain activation in prefrontal cortical regions in cocaine dependent subjects may be related to altered striatal dopamine function, and that enhancement of dopamine function via adenosine A2A receptor blockade could be explored further for amelioration of neurobehavioral deficits associated with chronic cocaine use.

  2. Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a Diverse Therapeutic Target: A Computational Perspective.

    Ramesh, M; Vepuri, Suresh B; Oosthuizen, Frasia; Soliman, Mahmoud E

    2016-02-01

    Adenosine monophosphate-activated protein kinase (AMPK) is viewed as a privileged therapeutic target for several diseases such as cancer, diabetes, inflammation, obesity, etc. In addition, AMPK has entered the limelight of current drug discovery with its evolution as a key metabolic regulator. AMPK also plays a key role in the maintenance of cellular energy homeostasis. Structurally, AMPK is a heterotrimeric protein, which consists of three protein subunits (α, β, and γ). The crystal structure of AMPK was solved, and several computational studies including homology modeling, molecular docking, molecular dynamics, and QSAR have been reported in order to explore the structure and function of this diverse therapeutic target. In this review, we present a comprehensive up-to-date overview on the computational and molecular modeling approaches that have been carried out on AMPK in order to understand its structure, function, dynamics, and its drug binding landscape. Information provided in this review would be of great interest to a wide pool of researchers involved in the design of new molecules against various diseases where AMPK plays a predominant role. Graphical Abstract ᅟ. PMID:26541160

  3. Drugs elevating extracellular adenosine administered in vivo induce serum colony-stimulating activity and interleukin-6 in mice

    Weiterová, Lenka; Hofer, Michal; Pospíšil, Milan; Znojil, V.; Štreitová, Denisa

    2007-01-01

    Roč. 56, č. 4 (2007), s. 463-473. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GP305/03/D050 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : extracellular adenosine * serum colony-stimulating activity * interleukin -6 Subject RIV: BO - Biophysics Impact factor: 1.505, year: 2007

  4. Recombinant Mouse PAP Has pH-Dependent Ectonucleotidase Activity and Acts through A1-Adenosine Receptors to Mediate Antinociception

    Sowa, Nathaniel A.; Kunjumon I. Vadakkan; Zylka, Mark J.

    2009-01-01

    Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A1-adenosine receptor (A1R) activation. In this study, we purified the secretory isoform of mouse (m)PAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mP...

  5. A role for adenosine deaminase in Drosophila larval development

    Doležal, T.; Doleželová, Eva; Žurovec, Michal; Bryant, P. J.

    2005-01-01

    Roč. 3, č. 7 (2005), s. 1213-1224. ISSN 1544-9173 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant ostatní: United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : Drosophila Subject RIV: ED - Physiology Impact factor: 14.672, year: 2005

  6. Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors.

    Yan, Xiao-Feng; Zhang, Zhong-Miao; Yao, Hong-Yi; Guan, Yan; Zhu, Jian-Ping; Zhang, Lin-Hui; Jia, Yong-Liang; Wang, Ru-Wei

    2013-11-01

    Mycelia of cultured Cordyceps sinensis (CS) is one of the most common substitutes for natural CS and was approved for arrhythmia in China. However, the role of CS in ameliorating injury during ischemia-reperfusion (I/R) is still unclear. We examined effects of extracts from CS on I/R and investigated the possible mechanisms. Post-ischemic coronary perfusion pressure, ventricular function, and coronary flow were measured using the Langendorff mouse heart model. Oxidative stress of cardiac homogenates was performed using an ELISA. Our results indicate that CS affords cardioprotection possibly through enhanced adenosine receptor activation. Cardioprotection was demonstrated by reduced post-ischemic diastolic dysfunction and improved recovery of pressure development and coronary flow. Treatment with CS largely abrogates oxidative stress and damage in glucose- or pyruvate-perfused hearts. Importantly, observed reductions in oxidative stress [glutathione disulfide (GSSG)]/[GSSG + glutathione] and [malondialdehyde (MDA)]/[superoxide dismutase + MDA] ratios as well as the resultant damage upon CS treatment correlate with functional markers of post-ischemic myocardial outcome. These effects of CS were partially blocked by 8-ρ-sulfophenyltheophylline, an adenosine receptor antagonist. Our results demonstrate a suppressive role of CS in ischemic contracture. Meanwhile, the results also suggest pre-ischemic adenosine receptor activation may be involved in reducing contracture in hearts pretreated with CS. PMID:23192916

  7. The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

    Sergio Zonszein

    Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

  8. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  9. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    Li, Hui-Guang [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Huang, Philip L. [American Biosciences, Boston, MA 02114 (United States); Zhang, Dawei; Sun, Yongtao [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Chen, Hao-Chia [Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892 (United States); Zhang, John [Department of Chemistry, New York University, New York, NY 10003 (United States); Huang, Paul L. [Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114 (United States); Kong, Xiang-Peng, E-mail: xiangpeng.kong@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Lee-Huang, Sylvia, E-mail: sylvia.lee-huang@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States)

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  10. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  11. Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

    Patel Shitalben R

    2009-06-01

    Full Text Available Abstract Background We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK and cytidine deaminase (CDA in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies. Methods Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of Results The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P Conclusion In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the

  12. Adenosine: front and center in linking nutrition and metabolism to neuronal activity

    Greene, Robert W

    2011-01-01

    Many individuals with epilepsy benefit from consuming a ketogenic diet, which is similar to the more commonly known Atkins diet. The underlying molecular reason for this has not been determined. However, in this issue of the JCI, Masino et al. have elucidated the mechanism responsible for the antiepileptic effects of the ketogenic diet in mice. The diet is shown to decrease expression of the enzyme adenosine kinase (Adk), which is responsible for clearing the endogenous antiepileptic agent ad...

  13. Adenosine can thwart antitumor immune responses elicited by radiotherapy. Therapeutic strategies alleviating protumor ADO activities

    Vaupel, Peter [Klinikum rechts der Isar, Technische Universitaet Muenchen (TUM), Department of Radiation Oncology, Munich (Germany); Multhoff, Gabriele [Klinikum rechts der Isar, Technische Universitaet Muenchen (TUM), Department of Radiation Oncology, Munich (Germany); Helmholtz Zentrum Muenchen, Institute for innovative Radiotherapy (iRT), Experimental Immune Biology, Neuherberg (Germany)

    2016-05-15

    By studying the bioenergetic status we could show that the development of tumor hypoxia is accompanied, apart from myriad other biologically relevant effects, by a substantial accumulation of adenosine (ADO). ADO has been shown to act as a strong immunosuppressive agent in tumors by modulating the innate and adaptive immune system. In contrast to ADO, standard radiotherapy (RT) can either stimulate or abrogate antitumor immune responses. Herein, we present ADO-mediated mechanisms that may thwart antitumor immune responses elicited by RT. An overview of the generation, accumulation, and ADO-related multifaceted inhibition of immune functions, contrasted with the antitumor immune effects of RT, is provided. Upon hypoxic stress, cancer cells release ATP into the extracellular space where nucleotides are converted into ADO by hypoxia-sensitive, membrane-bound ectoenzymes (CD39/CD73). ADO actions are mediated upon binding to surface receptors, mainly A2A receptors on tumor and immune cells. Receptor activation leads to a broad spectrum of strong immunosuppressive properties facilitating tumor escape from immune control. Mechanisms include (1) impaired activity of CD4 + T and CD8 + T, NK cells and dendritic cells (DC), decreased production of immuno-stimulatory lymphokines, and (2) activation of Treg cells, expansion of MDSCs, promotion of M2 macrophages, and increased activity of major immunosuppressive cytokines. In addition, ADO can directly stimulate tumor proliferation and angiogenesis. ADO mechanisms described can thwart antitumor immune responses elicited by RT. Therapeutic strategies alleviating tumor-promoting activities of ADO include respiratory hyperoxia or mild hyperthermia, inhibition of CD39/CD73 ectoenzymes or blockade of A2A receptors, and inhibition of ATP-release channels or ADO transporters. (orig.) [German] Untersuchungen des bioenergetischen Status ergaben, dass Tumorhypoxie neben vielen anderen bedeutsamen biologischen Effekten zu einem starken

  14. Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors.

    Carlin, Jesse Lea; Tosh, Dilip K; Xiao, Cuiying; Piñol, Ramón A; Chen, Zhoumou; Salvemini, Daniela; Gavrilova, Oksana; Jacobson, Kenneth A; Reitman, Marc L

    2016-02-01

    Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist-induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non-brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia. PMID:26606937

  15. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. PMID:27005940

  16. Endogenous activation of adenosine A(1) receptors accelerates ischemic suppression of spontaneous electrocortical activity

    Ilie, Andrei; Ciocan, Dragos; Zagrean, Ana-Maria;

    2006-01-01

    either 1.25 mg/kg DPCPX dissolved in 2 ml/kg dimethyl sulfoxide (DMSO) or the same volume of DMSO alone, 15 min before the third ischemic episode. Time to electrocortical suppression was estimated based on the decay of the root mean square of two-channel electrocorticographic recordings. During the first...... two ischemic episodes, electrocortical suppression appeared after approximately 12 s in both groups. After DMSO administration, ischemic suppression remained unchanged. After DPCPX administration, the time to electrocortical suppression was increased by approximately 10 s, and bursts of activity were...

  17. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  18. Absence epileptic activity changing effects of non-adenosine nucleoside inosine, guanosine and uridine in Wistar Albino Glaxo Rijswijk rats.

    Kovács, Z; Kékesi, K A; Dobolyi, Á; Lakatos, R; Juhász, G

    2015-08-01

    Adenosine (Ado) and non-adenosine (non-Ado) nucleosides such as inosine (Ino), guanosine (Guo) and uridine (Urd) may have regionally different roles in the regulation of physiological and pathophysiological processes in the central nervous system (CNS) such as epilepsy. It was demonstrated previously that Ino and Guo decreased quinolinic acid (QA)-induced seizures and Urd reduced penicillin-, bicuculline- and pentylenetetrazole (PTZ)-induced seizures. It has also been demonstrated that Ino and Urd may exert their effects through GABAergic system by altering the function of GABA(A) type of gamma-aminobutyric acid receptors (GABAA receptors) whereas Guo decreases glutamate-induced excitability through glutamatergic system, which systems (GABAergic and glutamatergic) are involved in pathomechanisms of absence epilepsy. Thus, we hypothesized that Ino and Guo, similarly to the previously described effect of Urd, might also decrease absence epileptic activity. We investigated in the present study whether intraperitoneal (i.p.) application of Ino (500 and 1000mg/kg), Guo (20 and 50mg/kg), Urd (500 and 1000mg/kg), GABA(A) receptor agonist muscimol (1 and 3mg/kg), GABA(A) receptor antagonist bicuculline (2 and 4mg/kg), non-selective Ado receptor antagonist theophylline (5 and 10mg/kg) and non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine maleate (MK-801, 0.0625 and 0.1250mg/kg) alone and in combination have modulatory effects on absence epileptic activity in Wistar Albino Glaxo Rijswijk (WAG/Rij) rats. We found that Guo decreased the number of spike-wave discharges (SWDs) whereas Ino increased it dose-dependently. We strengthened that Urd can decrease absence epileptic activity. Our results suggest that Guo, Urd and their analogs could be potentially effective drugs for treatment of human absence epilepsy. PMID:26037802

  19. Adenosine and Sleep

    Bjorness, Theresa E.; Greene, Robert W.

    2009-01-01

    Over the last several decades the idea that adenosine (Ado) plays a role in sleep control was postulated due in large part to pharmacological studies that showed the ability of Ado agonists to induce sleep and Ado antagonists to decrease sleep. A second wave of research involving in vitro cellular analytic approaches and subsequently, the use of neurochemical tools such as microdialysis, identified a population of cells within the brainstem and basal forebrain arousal centers, with activity t...

  20. Adenosine-Associated Delivery Systems

    Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

    2016-01-01

    Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

  1. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent ...

  2. Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and membrane transport.

    Kobayashi, S; Zimmermann, H; Millhorn, D E

    2000-02-01

    Acute exposure to hypoxia causes a release of adenosine (ADO) that is inversely related to the O2 levels in oxygen-sensitive pheochromocytoma (PC12) cells. In the current study, chronic exposure (48 h) of PC12 cells to moderate hypoxia (5% O2) significantly enhanced the release of ADO during severe, acute hypoxia (1% O2). Investigation into the intra- and extracellular mechanisms underpinning the secretion of ADO in PC12 cells chronically exposed to hypoxia revealed changes in gene expression and activities of several key enzymes associated with ADO production and metabolism, as well as the down-regulation of a nucleoside transporter. Decreases in the enzymatic activities of ADO kinase and ADO deaminase accompanied by an increase in those of cytoplasmic and ecto-5'-nucleotidases bring about an increased capacity to produce intra- and extracellular ADO. This increased potential to generate ADO and decreased capacity to metabolize ADO indicate that PC12 cells shift toward an ADO producer phenotype during hypoxia. The reduced function of the rat equilibrative nucleoside transporter rENT1 also plays a role in controlling extracellular ADO levels. The hypoxia-induced alterations in the ADO metabolic enzymes and the rENT1 transporter seem to increase the extracellular concentration of ADO. The biological significance of this regulation is unclear but is likely to be associated with modulating cellular activity during hypoxia. PMID:10646513

  3. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor.

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T N; Gregory, Karen J; Tosh, Dilip K; Christopoulos, Arthur; Jacobson, Kenneth A; May, Lauren T

    2016-07-01

    Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with significant N(6) or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5'-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  4. Activation of Adenosine Receptor A2A Increases HSC Proliferation and Inhibits Death and Senescence by Down-regulation of p53 and Rb

    Md. Kaimul eAhsan

    2014-04-01

    Full Text Available Background & Aims: During fibrosis hepatic stellate cells (HSC undergo activation, proliferation and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism.Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC.Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680 and blocked by a specific antagonist (ZM241385. By day twenty-one of culture primary rat HSC entered senescence and expressed -gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors.Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.

  5. Ultracytochemical localization of H+—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

    LUOSHENQIU; MASAHIROSAKAI; 等

    1990-01-01

    H-adenosine triphosphatase (H+-ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H+-ATPase,N-ethylmaleimide (NEM) or 4,4'-diisothiocyanostilbene-2,2'disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H+-ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H+-ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H+-ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H+-ATPase which may be derived from lysosomal membranes.

  6. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus.

    de Mendonça, A; Ribeiro, J A

    1997-08-01

    1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12-14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe. p.s.p.) recorded extracellularly from the stratum radiatum in the CAI area. 2. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2-50 nM), on the fe.p.s.p. slope (EC50 = 12.5 (9.2-17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 microM) (EC50 = 27.2 (21.4-34.5) nM, n = 4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 microM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). DHPG (10 microM) itself had an inhibitory effect of 20.1 +/- 1.9% (n = 4) on the fe.p.s.p. slope. 3. The concentration-response curves for the inhibitory effects of CPA (2-20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I; 1 microM), or in the presence of

  7. APOBEC deaminases-mutases with defensive roles for immunity

    Courtney; PROCHNOW; Ronda; BRANSTEITTER

    2009-01-01

    In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.

  8. Characterization of unexplored amidohydrolase enzyme-pterin deaminase.

    Jayaraman, Angayarkanni; Thandeeswaran, Murugesan; Priyadarsini, Ulaganathan; Sabarathinam, Shanmugam; Nawaz, K A Ayub; Palaniswamy, Muthusamy

    2016-06-01

    Pterin deaminase is an amidohydrolase enzyme hydrolyzing pteridines to form lumazine derivatives and ammonia. The enzyme captured the attention of scientists as early as 1959 and had been patented for its application as an anticancer agent. It is ubiquitously present in prokaryotes and has been reported in some eukaryotes such as honey bee, silkworm and rats. The enzyme has been observed to have a spectrum of substrates with the formation of respective lumazines. The role of the substrates of the enzyme in various metabolic pathways warrants a significant role in the biological activity of both prokaryotes and eukaryotes. Even though the functions of the enzyme have been explored in prokaryotes, their niche in the eukaryotic system is not clear. There is very few information on the structural and functional properties of the enzyme. This review has been congregated to emphasize the significance of pterin deaminase and analyzes the lacunae in understanding the biological characters of the enzyme. PMID:27094187

  9. AID/APOBEC cytosine deaminase induces genome-wide kataegis

    Lada Artem G

    2012-12-01

    Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

  10. Hydrogen potassium adenosine triphosphatase activity inhibition and downregulation of its expression by bioactive fraction DLBS2411 from Cinnamomum burmannii in gastric parietal cells

    Tjandrawinata RR; Nailufar F; Arifin PF

    2013-01-01

    Raymond R Tjandrawinata1,2, Florensia Nailufar2, Poppy F Arifin11Section of Molecular Pharmacology, 2Section of Animal Pharmacology, Dexa Medica, Cikarang, IndonesiaAbstract: This study assessed the gastric acid antisecretory effect of DLBS2411 fractionated from Cinnamomum burmannii. Hydrogen potassium adenosine triphosphatase (H+/K+ ATPase) activity and its gene expression were observed, and the antioxidant activity of DLBS2411 was also investigated. Treatment of DLBS2411 decreased the level...

  11. Triiodothyronine causes rapid reversal of alpha 1/cyclic adenosine monophosphate synergism on brown adipocyte respiration and type II deiodinase activity.

    Noronha, M; Raasmaja, A; Moolten, N; Larsen, P R

    1991-12-01

    Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between alpha 1- and beta-adrenergic pathways to stimulate type II iodothyronine 5'-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5'-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3'-triiodothyronine (T3). The fivefold synergism of alpha 1- and beta-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T3 treatment. Hypothyroidism reduced both the O2max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (10-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the alpha 1-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the alpha 1-agonist was lost within 2 days of T3 administration. These studies identify a second Ca(2+)-dependent intra-adrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (cAMP) responsiveness of the hypothyroid brown adipocyte. PMID:1683679

  12. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2.

    Peng, Shuang; Gerasimenko, Julia V; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Petersen, Ole H; Gerasimenko, Oleg V

    2016-08-01

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca(2+) signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca(2+) elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca(2+) signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5-10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca(2+) release followed by Ca(2+) entry and also substantially reduced Ca(2+) extrusion because of decreased intracellular ATP levels. The toxic Ca(2+) signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca(2+) signals and necrosis. We tested the effects of inhibiting the Ca(2+) release-activated Ca(2+) entry by the Ca(2+) channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca(2+) entry and also protected effectively against the development of necrosis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377732

  13. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels.

    Liu, Yumei; Zou, Haifeng; Zhao, Ping; Sun, Bo; Wang, Jinghua; Kong, Qingfei; Mu, Lili; Zhao, Sihan; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Zhao, Jiaying; Yin, Pengqi; Liu, Lei; Zhao, Xiuli; Li, Hulun

    2016-08-25

    Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4(+) T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca(2+)]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which may offer a therapeutic option for this disease. PMID:27217214

  14. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2

    Peng, Shuang; Gerasimenko, Julia V.; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Gerasimenko, Oleg V.

    2016-01-01

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca2+ signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca2+ elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca2+ signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5–10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. The toxic Ca2+ signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca2+ signals and necrosis. We tested the effects of inhibiting the Ca2+ release-activated Ca2+ entry by the Ca2+ channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca2+ entry and also protected effectively against the development of necrosis. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377732

  15. Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

    Macbeth, Mark R.; Lingam, Arunth T.; Bass, Brenda L.

    2004-01-01

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of on...

  16. Optogenetic Activation of Adenosine A2A Receptor Signaling in the Dorsomedial Striatopallidal Neurons Suppresses Goal-Directed Behavior.

    Li, Yan; He, Yan; Chen, Mozi; Pu, Zhilan; Chen, Li; Li, Ping; Li, Bo; Li, Haiyan; Huang, Zhi-Li; Li, Zhihui; Chen, Jiang-Fan

    2016-03-01

    The striatum has an essential role in neural control of instrumental behaviors by reinforcement learning. Adenosine A(2A) receptors (A(2A)Rs) are highly enriched in the striatopallidal neurons and are implicated in instrumental behavior control. However, the temporal importance of the A(2A)R signaling in relation to the reward and specific contributions of the striatopallidal A(2A)Rs in the dorsolateral striatum (DLS) and the dorsomedial striatum (DMS) to the control of instrumental learning are not defined. Here, we addressed temporal relationship and sufficiency of transient activation of optoA(2A)R signaling precisely at the time of the reward to the control of instrumental learning, using our newly developed rhodopsin-A2AR chimeras (optoA(2A)R). We demonstrated that transient light activation of optoA(2A)R signaling in the striatopallidal neurons in 'time-locked' manner with the reward delivery (but not random optoA(2A)R activation) was sufficient to change the animal's sensitivity to outcome devaluation without affecting the acquisition or extinction phases of instrumental learning. We further demonstrated that optogenetic activation of striatopallidal A(2A)R signaling in the DMS suppressed goal-directed behaviors, as focally genetic knockdown of striatopallidal A(2A)Rs in the DMS enhanced goal-directed behavior by the devaluation test. By contrast, optogenetic activation or focal AAV-Cre-mediated knockdown of striatopallidal A(2A)R in the DLS had relatively limited effects on instrumental learning. Thus, the striatopallidal A(2A)R signaling in the DMS exerts inhibitory and predominant control of goal-directed behavior by acting precisely at the time of reward, and may represent a therapeutic target to reverse abnormal habit formation that is associated with compulsive obsessive disorder and drug addiction. PMID:26216520

  17. Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

    Oliveira, L; Costa, A C; Noronha-Matos, J B; Silva, I; Cavalcante, W L G; Timóteo, M A; Corrado, A P; Dal Belo, C A; Ambiel, C R; Alves-do-Prado, W; Correia-de-Sá, P

    2015-02-01

    The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 μM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic

  18. Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail.

    Mittelstädt, Gerd; Moggré, Gert-Jan; Panjikar, Santosh; Nazmi, Ali Reza; Parker, Emily J

    2016-08-01

    Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. PMID:27191057

  19. Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

    Tello-Montoliu, Antonio; Thano, Estela; Rollini, Fabiana; Patel, Ronakkumar; Wilson, Ryan E; Muñiz-Lozano, Ana; Franchi, Francesco; Darlington, Andrew; Desai, Bhaloo; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2013-10-01

    Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (paspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (paspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators. PMID:23884248

  20. Comparison of the Effects of Adenosine A1 Receptors Activity in CA1 Region of the Hippocampus on Entorhinal Cortex and Amygdala Kindled Seizures in Rats

    A. Heidarianpour

    2008-10-01

    Full Text Available Introduction & Objective: In the CNS, adenosine is known to suppress repetitive neuronal Firing, suggesting a role as an endogenous modifier of seizures. Indeed, intracerebral adenosine concentrations rise acutely during seizure activity and are thought to be responsible for terminating seizures and establishing a period of post-ictal refractoriness. However, it is unclear whether this suppression results from a general depression of brain excitability or through action on particular sites critical for the control of after discharge generation and/or seizure development and propagation. In this regard, comparison of the effects of adenosine A1 receptors of CA1 (region of the ‎hippocampus on entorhinal cortex and amygdala kindled seizures was ‎investigated in this study. Materials & Methods: In this experimental study, Animals were kindled by daily electrical stimulation of amygdale (group A or entorhinal cortex (group B. In the fully kindled animals, N6-‎cyclohexyladenosine (CHA;1 and 10 M; a selective adenosine A1 receptor ‎agonist and 1,3-dimethyl-8-cyclohexylxanthine(CPT;1 ‎µ‎M; a selective ‎adenosine A1 receptors antagonist were microinfused bilaterally into the CA1 ‎region of hippocampus (1l/2min and animals were stimulated at 5 and 15 minutes after drug ‎injection. All animals were received artificial cerebrospinal fluid, 24 h before ‎each drug injection and this result were used as control. Results: The seizure parameters were measured at 5 and 15min post injection. Obtained data showed that CHA at concentrations of 10 ‎µ‎M reduced ‎entorhinal cortex and amygdala after discharge and stage5 seizure durations and ‎increased stage4 latency. CHA at concentration 1‎µ‎M significantly alters ‎seizure parameters of group A but not effect on group B. Intrahippocampal (CA1 region pretreatment of CPT (1 ‎µ‎M before CHA abolished the effects of CHA on seizure parameters.Conclusion: It ‎may be

  1. Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells

    Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [14C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [14C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [14C]aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [14C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased [14C]aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion

  2. Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficile infection in mice following vancomycin treatment

    Li Yuesheng

    2012-12-01

    Full Text Available Abstract Background Activation of the A2A adenosine receptor (A2AAR decreases production of inflammatory cytokines, prevents C. difficile toxin A-induced enteritis and, in combination with antibiotics, increases survival from sepsis in mice. We investigated whether A2AAR activation improves and A2AAR deletion worsens outcomes in a murine model of C. difficile (strain VPI10463 infection (CDI. Methods C57BL/6 mice were pretreated with an antibiotic cocktail prior to infection and then treated with vancomycin with or without an A2AAR agonist. A2AAR-/- and littermate wild-type (WT mice were similarly infected, and IFNγ and TNFα were measured at peak of and recovery from infection. Results Infected, untreated mice rapidly lost weight, developed diarrhea, and had mortality rates of 50-60%. Infected mice treated with vancomycin had less weight loss and diarrhea during antibiotic treatment but mortality increased to near 100% after discontinuation of antibiotics. Infected mice treated with both vancomycin and an A2AAR agonist, either ATL370 or ATL1222, had minimal weight loss and better long-term survival than mice treated with vancomycin alone. A2AAR KO mice were more susceptible than WT mice to death from CDI. Increases in cecal IFNγ and blood TNFα were pronounced in the absence of A2AARs. Conclusion In a murine model of CDI, vancomycin treatment resulted in reduced weight loss and diarrhea during acute infection, but high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. The administration of vancomycin plus an A2AAR agonist reduced inflammation and improved survival rates, suggesting a possible benefit of A2AAR agonists in the management of CDI to prevent recurrent disease.

  3. Overexpression of Activation-Induced Cytidine Deaminase in MTX- and Age-Related Epstein-Barr Virus-Associated B-Cell Lymphoproliferative Disorders of the Head and Neck

    Kentaro Kikuchi

    2015-01-01

    Full Text Available Recent research has shown that activation-induced cytidine deaminase (AID triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Such aberrant AID expression is seen in B-cell leukemia/lymphomas, including Burkitt lymphoma which is associated with c-myc translocation. Moreover, Epstein-Barr virus (EBV latent membrane protein-1 (LMP-1 increases genomic instability through early growth transcription response-1 (Egr-1 mediated upregulation of AID in B-cell lymphoma. However, few clinicopathological studies have focused on AID expression in lymphoproliferative disorders (LPDs. Therefore, we conducted an immunohistochemical study to investigate the relationship between AID and LMP-1 expression in LPDs (MTX-/Age-related EBV-associated, including diffuse large B-cell lymphomas (DLBCLs. More intense AID expression was detected in LPDs (89.5% than in DLBCLs (20.0%, and the expression of LMP-1 and EBER was more intense in LPDs (68.4% and 94.7% than in DLBCLs (10.0% and 20.0%. Furthermore, stronger Egr-1 expression was found in MTX/Age-EBV-LPDs (83.3% than in DLBCLs (30.0%. AID expression was significantly constitutively overexpressed in LPDs as compared with DLBCLs. These results suggest that increased AID expression in LPDs may be one of the processes involved in lymphomagenesis, thereby further increasing the survival of genetically destabilized B-cells. AID expression may be a useful indicator for differentiation between LPDs and DLBCLs.

  4. Perspectives of bacterial ACC deaminase in phytoremediation.

    Arshad, Muhammad; Saleem, Muhammad; Hussain, Sarfraz

    2007-08-01

    Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics. PMID:17573137

  5. Adenosine regulation of alveolar fluid clearance

    Factor, Phillip; Mutlu, Göskhan M.; Chen, Lan; Mohameed, Jameel; Akhmedov, Alexander T.; Meng, Fan Jing; Jilling, Tamas; Lewis, Erin Rachel; Johnson, Meshell D.; Xu, Anna; Kass, Daniel; Martino, Janice M.; Bellmeyer, Amy; Albazi, John S.; Emala, Charles

    2007-01-01

    Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na+ transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A1R), 2a (A2aR), 2b (A2bR), and 3 (A3R) adenosine receptors are present in rat and mouse lungs and alveol...

  6. Structural and biological function of NYD-SP15 as a new member of cytidine deaminases.

    Xu, Yidan; Li, Lei; Li, Jianmin; Liu, Qinghuai

    2016-05-25

    Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or DNA and were involved in the process of growth maintenance, host immunity, retroviral infection, tumorigenesis, and drug resistance with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth. PMID:26945630

  7. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems. PMID:27296962

  8. Adenosine: An immune modulator of inflammatory bowel diseases

    Jeff Huaqing Ye; Vazhaikkurichi M Rajendran

    2009-01-01

    Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation. Adenosine is transported via concentrative nucleoside transporter and equilibrative nucleoside transporter transporters that are localized in apical and basolateral membranes of intestinal epithelial cells, respectively. Increased extracellular adenosine levels activate the A2a receptor, which would reduce cytokines responsible for chronic inflammation.

  9. Adenosine (ADO) released during orthodromic stimulation of the frog sympathetic ganglion inhibits phosphatidylinositol turnover (PI) associated with synaptic transmission

    The authors have previously demonstrated that 3H-purine release was enhanced during synaptic activation of the prelabelled frog sympathetic ganglion. In addition, during orthodromic stimulation, there is an increased 3H-inositol release (an index of PI) that occurs during the poststimulation period and not during the period of stimulation. They hypothesized that endogenous ADO inhibits PI turnover during orthodromic stimulation. To test this hypothesis (1) they performed experiments to directly measure ADO release in the extracellular fluid by placing the ganglion in a 5 μl drop of Ringer's and let it come to equilibrium with the interstitial fluid, (2) they destroyed endogenous ADO by suffusing adenosine deaminase (ADA) during the stimulation period. Their results show (1) orthodromic stimulation increases release of ADO into the bathing medium, (2) ADA induced an increase of PI during the stimulation period in contrast to an increase seen only during the poststimulation period when ADA was omitted. They conclude that there is dual control of PI during synaptic activity, a stimulatory effect (cause unknown) and a short lived inhibitory effect that is probably caused by adenosine

  10. Targeting energy metabolic and oncogenic signaling pathways in triple-negative breast cancer by a novel adenosine monophosphate-activated protein kinase (AMPK) activator.

    Lee, Kuen-Haur; Hsu, En-Chi; Guh, Jih-Hwa; Yang, Hsiao-Ching; Wang, Dasheng; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2011-11-11

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC(50), 0.3 μM) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC(50), 5 and 2 μM, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47-49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  11. Non-redundancy of cytidine deaminases in class switch recombination.

    Fugmann, Sebastian D; Rush, James S; Schatz, David G

    2004-03-01

    Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism. PMID:14991614

  12. Adenosine in exercise adaptation.

    Simpson, R E; Phillis, J. W.

    1992-01-01

    By influencing the regulation of the mechanisms of angiogenesis, erythropoietin production, blood flow, myocardial glucose uptake, glycogenolysis, systolic blood pressure, respiration, plasma norepinephrine and epinephrine levels, adenosine may exert a significant effect on the body's adaptation response to exercise. However, adenosine's possible influence over the vasodilatory response to exercise in skeletal muscle is controversial and more research is required to resolve this issue. Variou...

  13. Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases

    AtsushiKoito

    2013-02-01

    Full Text Available Over 40% of the human genome is recognizable as having been derived from ancient retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, with an additional few percent classified as DNA transposable elements. Endogenous retroviruses are long terminal repeat (LTR-type retroelements that account for ~8% of human genomic DNA. Non-LTR members are present at extremely high copy numbers, with ~17% of the human genome consisting of long interspersed nuclear elements (LINEs. These LINEs modify vertebrate genomes not only through insertions, but also by the indirect replication of nonautonomous retrotransposons, such as short interspersed nuclear elements. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that confer beneficial genetic diversity and those that cause deleterious gene disruptions. The mammalian cytidine deaminases encoded by the APOBEC3 genes can restrict a broad number of exogenous pathogens, such as exogenous retroviruses, and the mobility of endogenous retroelements. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the cytidine (C to uridine (U deamination of apolipoprotein B mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian APOBEC cytidine deaminases, which can bind to single-stranded DNA (ssDNA as well as RNA, are able to insert mutations into ssDNA and/or RNA as a result of their ability to deaminate C to U. While these APOBEC cytidine deaminases with DNA mutagenic activity can be deleterious to cells, their biological modifications, such as protein-protein interactions and subcellular localization, in addition to their ability to bind to RNA, appear to have conferred a role for APOBECs as a cellular defense system against retroviruses and retroelements. In support of this notion, the expansion of the single APOBEC3 gene in mice to the seven

  14. Role of adenosine receptors in caffeine tolerance

    Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity of caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity

  15. Macrophages increase the resistance of pancreatic adenocarcinoma cells to gemcitabine by upregulating cytidine deaminase

    Amit, Moran; Gil, Ziv

    2013-01-01

    Tumor-associated macrophages play a central role in tumor progression and metastasis. Macrophages can also promote the resistance of malignant cells to chemotherapy by stimulating the upregulation of cytidine deaminase, an intracellular enzyme that catabolizes the active form of gemcitabine. Targeting macrophage-dependent chemoresistance may reduce tumor-associated morbidity and mortality. PMID:24498570

  16. Effect of the growth stage and cultivar on policosanol profiles of barley sprouts and their adenosine 5'-monophosphate-activated protein kinase activation.

    Seo, Woo Duck; Yuk, Heung Joo; Curtis-Long, Marcus J; Jang, Ki Chang; Lee, Jin Hwan; Han, Sang-Ik; Kang, Hang Won; Nam, Min Hee; Lee, Sung-Joon; Lee, Ji Hae; Park, Ki Hun

    2013-02-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an intracellular sensor that can regulate glucose levels within the cell. For this reason, it is well-known to be a target for drugs against diabetes and obesity. AMPK was activated significantly by the hexane extract of barley sprouts. This AMPK activation emerges across the growth stages of the sprout, becoming most significant (3 times above the initial stages) 10 days after sprouting. After this time, the activation decreased between 13 and 20 days post-sprouting. Analysis of the hexane extracts by gas chromatography-mass spectrometry showed that the amounts of policosanols (PCs, which are linear, primary aliphatic alcohols with 20-30 carbons) in the plant dramatically increased between 5 days (109.7 mg/100 g) and 10 days (343.7 mg/100 g) post-sprouting and then levels fell back down, reaching 76.4 mg/100 g at 20 days post-sprouting. This trend is consistent with PCs being the active ingredient in the barley plants. We validate this by showing that hexacosanol is an activator of AMPK. The richest cultivar for PCs was found to be the Daejin cultivar. Cultivars had a significant effect on the total PC content (113.2-183.5 mg/100 g) within the plant up to 5 days post-sprouting. However this dependence upon the cultivar was not so apparent at peak stages of PC production (10 days post-sprouting). The most abundant PC in barley sprout, hexacosanol, contributed 62-80% of the total PC content at every stage. These results are valuable to determine the optimal times of harvest to obtain the highest yield of PCs. PMID:23301834

  17. Study the effect of bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase on resistance to salt stress in tomato plant

    Maryam SADRNIA

    2011-11-01

    Full Text Available 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase produced by rhizobacteria could be remove theethylene precursor and stimulate plant growth. Aim of the work was investigation on effect of rhizosphere bacteria Pseudomonasmendocina containing plasmid carrying gene encoding ACC deaminase on resistance of tomato plant to salinity. Amplification ofacds gene in selected Pseudomonas was performed; the g e n e w a s c l o n e d i n Escherichia coli and was cloned subsequently in P.mendocina. Enzyme activity was determined in cloned Escherichia coli and cloned P. mendocina for confirmation of geneexpression. Effect of bacterial ACC deaminase on resistance of tomato plants to NaCl was studied in Pot and Greenhouse. In potexperiment, tomato plant treated by cloned P. mendocina was compared with plants treated by P. mendocina (without plasmid andcontrol group. Salinity were established by adding 172 and 207 mM of NaCl to irrigated water. Greenhouse experiments wereconducted in similar groups of bacteria in 207 mM of NaCl. Results obtained from pot experiment revealed that plants treated bycloned P. mendocina in 172 mM of NaCl was showed increasing content of growth than ones treated by P. mendocina and controlas 11%, 18.4% growth for the shoot, 16.6%, 3.7% for roots and 9.6%, 27.5% for wet weight after five weeks, respectively. In 207mM of NaCl, the results were as 14.9 %, 9.7% for shoot, 94.3%, 15.7% for roots and 96.4%, 50.6% for wet weight, respectively. Ingreenhouse experiment, results in same parameter in 207 mM of NaCl were revealed as 63.7%, 7 times for shoot, 2.8, 14 times forroots and 66.1%, 154 times for wet weight, respectively. We concluded that recombinant P. mendocina producing ACC deaminaseby reduction of ethylene content of tomato plant in high salt concentrations could result in improvement of plant resistance tosalinity.

  18. 腺苷脱氨酶、C型凝集素及丝氨酸蛋白酶抑制剂基因在白纹伊蚊唾液腺中的表达%Expression of the Genes of Adenosine Deaminase, C-lectin and Serpin in the Salivary Gland of Aedes albopictus

    吴家红; 程金芝; 陈璐; 孙宇

    2010-01-01

    目的 了解腺苷脱氨酶(adenosine deal-flinase,ADA)基因、C型凝集素(C-lectin)基因和丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)基因在白纹伊蚊唾液腺中的表达情况.方法 分别制备未吸血雌蚊唾液腺(SG组) 和吸血雌蚊唾液腺(BSG组)、雌蚊躯体(无头无唾液腺,C组)和雄蚊组织(无头含唾液腺,M组)的总RNA,采用实时荧光定量RT.PCR技术,以β肌动蛋白(β-actin)基因作为内参,根据GenBank公布的ADA、C-lectin和Serpin基因序列设计特异性引物进行RT-PCR.分析以上基因在不同组织中的表达情况.结果 与雌蚊躯体和雄蚊组织中相对表达量相比,ADA基因在雌蚊唾液腺中的表达分别提高了545倍和123倍(P<0.01);C-lectin基因在雌蚊唾液腺中的表达分别提高了3 929倍和4 973倍(P<0.01);Serpin基因在雌蚊唾液腺中的表达分别提高了1 911倍和2 978倍(P<0.01).吸血前后雌蚊唾液腺中ADA、C-lectin和Serpin等3个基因的表达差异无统计学意义(P>0.05).结论 ADA在蚊虫组织中均有表达,在雌蚊唾液腺中高表达;C-lectin和serpin基因在雌蚊唾液腺中特异高表达.

  19. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants. PMID:25674802

  20. Aldehyde dehydrogenase type 2 activation by adenosine and histamine inhibits ischemic norepinephrine release in cardiac sympathetic neurons: mediation by protein kinase Cε.

    Robador, Pablo A; Seyedi, Nahid; Chan, Noel Yan-Ki; Koda, Kenichiro; Levi, Roberto

    2012-10-01

    During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde- and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase Cε (PKCε). Selective activation of G(i/o)-coupled adenosine A(1), A(3), or histamine H(3) receptors markedly inhibited both acetaldehyde- and hypoxia-induced norepinephrine release. These effects were also abolished by PKCε and/or ALDH2 inhibition. Moreover, A(1)-, A(3)-, or H(3)-receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H(3) receptors). This action was prevented by the inhibition of PKCε and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A(1)-, A(3)-, and H(3)-receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKCε and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKCε and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that

  1. A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

    3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

  2. Adenosine Receptors and Asthma

    Wilson, Constance N; Nadeem, Ahmed; Spina, Domenico; Brown, Rachel; Page, Clive P.; Jamal Mustafa, S.

    2009-01-01

    The pathophysiological processes underlying respiratory diseases like asthma are complex, resulting in an overwhelming choice of potential targets for the novel treatment of this disease. Despite this complexity, asthmatic subjects are uniquely sensitive to a range of substances like adenosine, thought to act indirectly to evoke changes in respiratory mechanics and in the underlying pathology, and thereby to offer novel insights into the pathophysiology of this disease. Adenosine is of partic...

  3. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems

    Iyer, Lakshminarayan M; Zhang, Dapeng; Rogozin, Igor B.; Aravind, L.

    2011-01-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains ar...

  4. Theophylline and adenosine modulate the inflammatory functions of the human neutrophil by exerting an opposing influence on the stimulus-induced increase in intracellular calcium

    Based on evidence that endogenously-produced adenosine inhibited neutrophil responses, the influence of methylxanthine bronchodilators on neutrophil responses stimulated in vitro by n-formyl-methionyl-leucyl-phenylalanine (fMLP) was examined. At concentrations between 10/sup /minus/5/ M and 10/sup /minus/4/ M, theophylline potentiated lysosomal enzyme release by 30 to 50%, superoxide anion formation by 30 to 60%, and neutrophil aggregation. Theophylline at concentrations >10/sup /minus/4/ M inhibited the same responses by >90%. Adenosine deaminase mimicked, whereas adenosine reversed the theophylline potentiation. A potential role for calcium in the modulation of the neutrophil responses by theophylline and adenosine was explored. Theophylline enhanced by >150% the fMLP-stimulated increase in cytoplasmic calcium concentration ([Ca2+]/sub i/) at time points between 5 and 90 sec as measured by Fura-2. Adenosine deaminase induced a comparable enhancement, whereas 3 /times/ 10/sup /minus/7/ M adenosine and 10/sup /minus/7/ M N-ethylcarboxamideadenosine decreased the [Ca2+]/sub i/ in fMLP-stimulated neutrophils. Extracellular calcium was not required for the opposing influences of theophylline and adenosine and neither compound altered fMLP-stimulated 45Ca uptake at the early time points

  5. Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: A structural and pre-steady state kinetic analysis

    Siggaard, Julie; Johansson, Eva; Vognsen, Tina Reinholdt; Helt, Signe Smedegaard; Harris, Pernille; Larsen, Sine; Willemoës, Martin

    2009-01-01

    Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had...... nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar...

  6. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels.

    Proks, Peter; Puljung, Michael C; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M

    2016-08-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues-mainly intracellular adenine nucleotide concentrations-to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377720

  7. Attenuation of gastric mucosal inflammation induced by aspirin through activation of A2A adenosine receptor in rats

    Masaru Odashima; Reina Ohba; Sumio Watanabe; Joel Linden; Michiro Otaka; Mario Jin; Koga Komatsu; Isao Wada; Youhei Horikawa; Tamotsu Matsuhashi; Natsumi Hatakeyama; Jinko Oyake

    2006-01-01

    AIM: To determine whether a specific adenosine A2A receptor agonist (ATL-146e) can ameliorate aspirin-induced gastric mucosal lesions in rats, and reduce neutrophil accumulation and production of pro-inflammatory cytokines.METHODS: Gastric lesions were produced by oral gavage of aspirin (200 mg/kg) and HCl (0.15 mol/L,8.0 mL/kg). 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL-146e,2.5-5 μg/kg, IP) was injected 30 min before the administration of aspirin. Tissue myeloperoxidase (MPO) concentration in gastric mucosa was measured as an index of neutrophil infiltration. Gastric mucosal concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined by ELISA. Also, we examined the effect of ATL-146e on tissue prostaglandin E2 (PGE2) production and gastric secretion.RESULTS: Intragastric administration of aspirin induced multiple hemorrhagic erosions in rat gastric mucosa. The total length of gastric erosions (ulcer index) in control rats was 29.8±7.75 mm and was reduced to 3.8±1.42 mm after pretreatment with 5.0 g/kg ATL-146e (P< 0.01).The gastric contents of MPO and pro-inflammatory cytokines were all increased after the administration of aspirin and reduced to nearly normal levels by ATL-146e.Gastric mucosal PGE2 concentration was not affected by intraperitoneal injection of ATL-146e.CONCLUSION: The specific adenosine A2A receptor agohist, ATL-146e, has potent anti-ulcer effects presumably mediated by its anti-inflammatory properties.

  8. Effect of adenosine and adenosine analogs on ( sup 14 C)aminopyrine accumulation by rabbit parietal cells

    Ota, S.; Hiraishi, H.; Terano, A.; Mutoh, H.; Kurachi, Y.; Shimada, T.; Ivey, K.J.; Sugimoto, T. (Univ. of Tokyo (Japan))

    1989-12-01

    Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on (14C)aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. (14C)Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated (14C)aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on (14C)aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased (14C)aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

  9. Alternative transcription and splicing of the human porphobilinogen deaminase gene

    Porphobilinogen deaminase is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. The authors have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. They demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the β-globin gene promoters

  10. Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat

    V Haktan Ozacmak; Hale Sayan

    2007-01-01

    AIM: To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury.METHODS: Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N6-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase,malondialdehyde, and reduced glutathione levels were measured.RESULTS: Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist.CONCLUSION: The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

  11. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment

    Ohta, Akio

    2016-01-01

    Within tumors, some areas are less oxygenated than others. Since their home ground is under chronic hypoxia, tumor cells adapt to this condition by activating aerobic glycolysis; however, this hypoxic environment is very harsh for incoming immune cells. Deprivation of oxygen limits availability of energy sources and induces accumulation of extracellular adenosine in tumors. Extracellular adenosine, upon binding with adenosine receptors on the surface of various immune cells, suppresses pro-inflammatory activities. In addition, signaling through adenosine receptors upregulates a number of anti-inflammatory molecules and immunoregulatory cells, leading to the establishment of a long-lasting immunosuppressive environment. Thus, due to hypoxia and adenosine, tumors can discourage antitumor immune responses no matter how the response was induced, whether it was spontaneous or artificially introduced with a therapeutic intention. Preclinical studies have shown the significance of adenosine in tumor survival strategy by demonstrating tumor regression after inactivation of adenosine receptors, inhibition of adenosine-producing enzymes, or reversal of tissue hypoxia. These promising results indicate a potential use of the inhibitors of the hypoxia–adenosine pathway for cancer immunotherapy. PMID:27066002

  12. Adenosine stress protocols for myocardial perfusion imaging

    Baškot Branislav

    2008-01-01

    Full Text Available Background/Aim. Treadmill test combined with myocardial perfusion scintigraphy (MPS is a commonly used technique in the assessment of coronary artery disease. There are many patients, however, who may not be able to undergo treadmill test. Such patients would benefit from pharmacological stress procedures combined with MPS. The most commonly used pharmacological agents for cardiac stress are coronary vasodilatators (adenosine, dipyridamol and catecholamines. Concomitant low-level treadmill exercise with adenosine pharmacologic stress (AdenoEX during MPS has become commonly used in recent years. A number of studies have demonstrated a beneficial impact of AdenoEX protocol. The aim of the study was, besides introducing into practice the two types of protocols of pharmatological stress test with adenosine, as a preparation for MPS, to compare and monitor the frequency of their side effects to quality, acquisition, as well as to standardize the onset time of acquisition (diagnostic imaging for both protocols. Methods. A total of 130 patients underwent pharmacological stress test with adenosine (vasodilatator. In 108 of the patients we performed concomitant exercise (AdenoEX of low level (50W by a bicycle ergometar. In 28 of the patients we performed Adenosine abbreviated protocol (AdenoSCAN. Side effects of adenosine were followed and compared between the two kinds of protocols AdenoEX and AdenoSCAN. Also compared were image quality and suggested time of acquisition after the stress test. Results. Numerous side effects were found, but being short-lived they did not require any active interventions. The benefit of AdenoEX versus AdenoSCAN included decreased side effects (62% vs 87%, improved safety and patients tolerance, improved target-to-background ratios because of less subdiaphragmatic activity, earlier acquisition, and improved sensitivity. Conclusion. The safety and efficacy of adenosine pharmacological stress is even better with concomitant

  13. Turnover of adenosine in plasma of human and dog blood

    To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a ''stopping solution.'' Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [3H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole

  14. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors. PMID:25687775

  15. Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia-induced vasodilatation

    Frøbert, Ole; Haink, Gesine; Simonsen, Ulf; Gravholt, Claus H; Levin, Max; Deussen, Andreas

    2006-01-01

    We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F2α (10−5m), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [14C]adenosine over the microdialysis membrane increased from 0.32 ± 0.02 to 0.46 ± 0.02 (n = 4, P < 0.01) during the study period. At baseline, interstitial adenosine was in the region of 10 nm which is significantly less than previously found myocardial concentrations. Hypoxia (PO2 30 mmHg for 60 min, n = 5) increased coronary diameters by 20.0 ± 2.6% (versus continuous oxygenation −3.1 ± 2.4%, n = 6, P < 0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with erythro-9-(2-hydroxy-3-nonyl-)-adenine, 5 μm), adenosine kinase (with iodotubericidine, 10 μm) and adenosine transport (with n-nitrobenzylthioinosine, 1 μm) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilatation similar to that during hypoxia could be obtained with 30 μm of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n = 5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration–response experiments showed vasodilatation to be more pronounced during hypoxia (n = 9) than during normoxia (n = 9, P < 0.001) and the A2A receptor antagonist ZM241385 (20 nm, n = 5), attenuated hypoxia-induced vasodilatation while the selective A2B receptor antagonist MRS1754 (20 nm, n = 4), had no effect. The lactate/pyruvate ratio was significantly increased in

  16. Mechanism of protection of adenosine from sulphate radical anion and repair of adenosine radicals by caffeic acid in aqueous solution

    M Sudha Swaraga; L Charitha; M Adinarayana

    2005-07-01

    The photooxidation of adenosine in presence of peroxydisulphate (PDS) has been studied by spectrophotometrically measuring the absorbance of adenosine at 260 nm. The rates of oxidation of adenosine by sulphate radical anion have been determined in the presence of different concentrations of caffeic acid. Increase in [caffeic acid] is found to decrease the rate of oxidation of adenosine suggesting that caffeic acid acts as an efficient scavenger of $SO_{4}^{\\bullet-}$ and protects adenosine from it. Sulphate radical anion competes for adenosine as well as for caffeic acid. The quantum yields of photooxidation of adenosine have been calculated from the rates of oxidation of adenosine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to sulphate radical anion. From the results of experimentally determined quantum yields (exptl) and the quantum yields calculated (cal) assuming caffeic acid acting only as a scavenger of $SO_{4}^{\\bullet-}$ show that exptl values are lower than cal values. The ' values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for $SO_{4}^{\\bullet-}$ scavenging by caffeic acid, are also found to be greater than exptl values. These observations suggest that the transient adenosine radicals are repaired by caffeic acid in addition to scavenging of sulphate radical anions.

  17. Effect of theophylline on adenosine production in the canine myocardium

    Adenosine is thought to participate in local regulation of coronary blood flow. However, competitive antagonists of adenosine fail to block myocardial active hyperemia. The authors examined the effect of locally administered theophylline on active hyperemia and myocardial adenosine production during intracoronary isoproterenol infusion in the dog heart. Isoproterenol decreased coronary resistance and increased myocardial adenosine production. Infusion of theophylline at a rate that attenuated the vasodilator response to exogenously administered adenosine failed to attenuate the increase in coronary blood flow produced by isoproterenol. However, theophylline plus isoproterenol production greater increases in myocardial adensine production than isoproterenol alone. The curves relating resistance and adenosine in the presence of theophylline fell to the right of those in the absence of theophylline. These findings suggest that the failure of theophylline to attenuate isoproterenol hyperemia in the dog heart results at least in part from an increase in adenosine concentration at the arteriole to a level beyond that blocked by this competitive antagonist and that adenosine may in fact play a role in isoproterenol-induced active hyperemia

  18. Adenosine and sleep

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A1 receptors, 3H-L-PIA binding was measured. The Bmax values for 3H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in 3H-L-PIA binding resulted from REM sleep deprivation and not from stress

  19. Adenosine and sleep

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  20. Cytotoxic purine nucleoside analogues bind to A1, A2A and A3 adenosine receptors

    Jensen, Kyle; Johnson, L’Aurelle A.; Jacobson, Pamala A.; Kachler, Sonja; Kirstein, Mark N.; Lamba, Jatinder; Klotz, Karl-Norbert

    2012-01-01

    Fludarabine, clofarabine and cladribine are anti-cancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A1, A2A, A2B, and A3). Radioligand bindin...

  1. Adenosine as a signaling molecule in the retina: biochemical and developmental aspects

    ROBERTO PAES-DE-CARVALHO

    2002-01-01

    The nucleoside adenosine plays an important role as a neurotransmitter or neuromodulator in the central nervous system, including the retina. In the present paper we review compelling evidence showing that adenosine is a signaling molecule in the developing retina. In the chick retina, adenosine transporters are present since early stages of development before the appearance of adenosine A1 receptors modulating dopamine-dependent adenylate cyclase activity or A2 receptors that directly activa...

  2. Identification of small molecule compounds with higher binding affinity to guanine deaminase (cypin) than guanine

    Fernández, José R.; Sweet, Eric S; Welsh, William J.; Firestein, Bonnie L

    2010-01-01

    Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transp...

  3. Pleural effusion: Role of pleural fluid cytology, adenosine deaminase level, and pleural biopsy in diagnosis

    Biswajit Biswas

    2016-01-01

    Full Text Available Objective: The present study is designed to evaluate the role of pleural fluid analysis in diagnosing pleural diseases and to study the advantages and disadvantages of thoracocentasis and pleural biopsy. Materials and Methods: We prospectively included 66 consecutive indoor patients over a duration of 1 year. Pleural fluid was collected and cytological smears were made from the fluid. Plural biopsy was done in the same patient by Cope needle. Adequate pleural biopsy tissue yielding specific diagnosis was obtained in 47 (71.2% cases. Results: Tuberculosis was the commonest nonneoplastic lesion followed by chronic nonspecific pleuritis comprising 60% and 33.3% of the nonneoplastic cases respectively and tuberculosis was predominantly diagnosed in the younger age group. Majority (70.8% of malignancy cases were in the age group of >50-70. Adenocarcinoma was found to be the commonest (66.7% malignant neoplasm in the pleurae followed by small-cell carcinoma (20.8%. Conclusion: Pleural biopsy is a useful and minimally invasive procedure. It is more sensitive and specific than pleural fluid smears.

  4. Two Cases of Severe Combined Immunodeficiency Caused By Adenosine Deaminase Deficiency

    Turkan Patiroglu

    2014-08-01

    Full Text Available Severe Combined Immune Deficiency (SCID is a primary immune deficiency disorder manifested with severe infections upon first months of life, which is characterized by diverse genetic defects in T and B lymphocyte functions and occasionally in NK cells. ADA deficiency is a form of SCID progressing with severe lymphopenia and immune deficiency caused by toxic metabolites of ADA. Bone marrow transplantation (BMT is the only curative treatment although prophylactic anti-microbial therapy, intravenous immunoglobulin (IVIG and enzyme replacement can achieve transient improvements. Early diagnosis before development of severe infections and organ injury and referral to pediatric immunology clinics will make considerable contributions to prognosis. Here, we presented 2 cousins with SCID who had positive family history with deceased sibling; presented with tanning at skin, severe neonatal infections and Q246X (c736C>T non-sense mutation in exon 8 in ADA gene  in order to emphasize this rare mutation and pediatric emergencies associated with this disorder.

  5. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. PMID:23598277

  6. Oxidative stress parameters and erythrocyte membrane adenosine triphosphatase activities in streptozotocin-induced diabetic rats administered aqueous preparation of Kalanchoe Pinnata leaves

    Nikhil Menon

    2016-01-01

    Full Text Available Background: Diabetes mellitus is a chronic metabolic disease that according to the World Health Organization affects more than 382 million people. The rise in diabetes mellitus coupled with the lack of an effective treatment has led many to investigate medicinal plants to identify a viable alternative. Objective: To evaluate red blood cell (RBC membrane adenosine triphosphatase (ATPase activities and antioxidant levels in streptozotocin-induced diabetic rats administered aqueous preparation of Kalanchoe pinnata leaves. Materials and Methods: Diabetes mellitus was induced in rats by a single administration of streptozotocin (60 mg/kg. Diabetic rats were then treated with aqueous K. pinnata preparation (three mature leaves ~ 9.96 g/70 kg body weight or about 0.14 g/kg body weight/day for 30 days. Serum glucose, RBC membrane ATPase activities, and antioxidant levels were determined. Results: We noted weight loss and reduced food consumption in the treated diabetic group. Serum glucose levels were reduced in the treated diabetic group compared to the other groups. Superoxide dismutase activity and glutathione levels were not significantly elevated in the treated group compared to the diabetic group. However, serum catalase activity was significantly (P < 0.05 increased in the treated diabetic group compared to the other groups. Serum thiobarbituric acid reactive substances were not significantly altered among the groups. There was a significant (P < 0.05 increase in Mg2+ ATPase activity and a nonsignificant increase in Na+/K+ ATPase activity in the RBC membrane of the treated diabetic group compared to the diabetic group. Conclusion: The consumption of aqueous preparation of K. pinnata may accrue benefits in the management of diabetes by lowering oxidative stress often associated with the disease and improving the availability of cellular magnesium through an increase in the magnesium ATPase pump in the RBC membrane for increased cellular metabolism

  7. Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

    Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site

  8. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong

    2011-01-01

    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  9. Effect of an antioxidant combination on the distribution of acetylcholinesterase and adenosine triphosphatase activities in the cerebellum of in lindane-intoxicated mice

    Devendra Kumar Bhatt

    2013-04-01

    Full Text Available Objective: The present investigation ascertains a protective potential of a combination of antioxidants against lindane-induced toxicity in cerebellum of mice. Methods: For the study, animals are divided into four groups. First group is control and it is given only vehicle. Second group is treated with lindane and analysed if there are any lesions in the brain. Third group is purely antioxidants treated group; four antioxidants, i.e. resveratrol, ascorbic acid, alpha lipoic acid and vitamin E, are subcutaneously administered in a suitable combination to the animals of this group. Fourth group is treated with both lindane and antioxidants. Acetylcholinesterase (AChE and adenosine triphosphatase (ATPase activities are used as histochemical markers for manifestation of lindane-induced acute toxicity. Biochemical levels of glutathione (GSH and thiobarbituric acid reactive substances (TBARS were also evaluated for different groups to confirm the toxicity of lindane in cerebellum. Results: Treatment with lindane caused decrease in AChE and ATPase activities, and GSH levels in cerebellum whereas a significant increase was recorded in TBARS. Antioxidants treatment increased the enzymatic activities. A significant rise in GSH level was recorded in the antioxidant treatment group as compared to group I and group II whereas TBARS levels were significantly reduced. GSH and TBARS levels altered significantly in group IV as compared to control group and lindane-treated group. In group III, AChE and ATPase activities increased in layers and nuclei of cerebellum as compared to control group. Conclusions: Since the use of antioxidants prevents the inhibition of AChE and ATPase, functions which are altered due to lindane-toxicity may be protected. [J Exp Integr Med 2013; 3(2.000: 103-112

  10. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique. PMID:25983132