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1.
Chem Biol Interact ; 311: 108796, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421116

RESUMO

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5'-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1-3 and 5'-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin. Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.


Assuntos
Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nitrilos/farmacologia , Nucleotidases/genética , Nucleosídeos de Purina/metabolismo , Piretrinas/farmacologia , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Masculino , Espectrometria de Massas , Nitrilos/sangue , Nitrilos/metabolismo , Nucleotidases/metabolismo , Piretrinas/sangue , Piretrinas/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Ratos Wistar
2.
Mol Cell Endocrinol ; 479: 54-60, 2019 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-30184475

RESUMO

The incidence of papillary thyroid carcinoma (PTC) has been increasing, which raised the interest in its molecular pathways. Although the high expression of ecto-5'-nucleotidase (NT5E) gene expression and NT5E enzymatic activity in several types of cancer is associated with tumor progression, its role in PTC remains unknown. Here, we investigated the AMP hydrolysis in human normal thyroid cells and PTC cells, in primary culture, and the association of NT5E expression with clinical aspects of PTC patients. AMPase activity was higher in thyroid cells isolated from PTC, as compared to normal thyroid (P = 0.0063). Significant correlation was observed between AMPase activity and NT5E levels in primary thyroid cell cultures (r = 0.655, P = 0.029). NT5E expression was higher in PTC than in the adjacent non-malignant thyroid tissue (P = 0.0065) and were positively associated with metastatic lymph nodes (P = 0.0007), risk of recurrence (P = 0.0033), tumor size (P = 0.049), and nodular hyperplasia in the adjacent thyroid parenchyma, when compared to normal thyroid or lymphocytic thyroiditis (P = 0.0146). After adjusting for potential confounders, the malignant/non-malignant paired expression ratio of NT5E mRNA was independently associated with metastatic lymph nodes (P = 0.0005), and tumor size (P=0.0005). In addition, the analysis of PTC described in the TCGA database also showed an association between higher expression of NT5E and metastatic lymph nodes, and tumor microinvasion. These results support the hypothesis that NT5E have a role in PTC microenvironment and might be a potential target for PTC therapy.


Assuntos
5'-Nucleotidase/metabolismo , Linfonodos/enzimologia , Linfonodos/patologia , Metástase Linfática/patologia , Câncer Papilífero da Tireoide/enzimologia , Câncer Papilífero da Tireoide/patologia , 5'-Nucleotidase/genética , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Nucleotidases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Câncer Papilífero da Tireoide/genética , Glândula Tireoide/patologia
3.
J Immunol Res ; 2018: 4892473, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30159340

RESUMO

Evidences show that purinergic signaling is involved in processes associated with health and disease, including noncommunicable, neurological, and degenerative diseases. These diseases strike from children to elderly and are generally characterized by progressive deterioration of cells, eventually leading to tissue or organ degeneration. These pathological conditions can be associated with disturbance in the signaling mediated by nucleotides and nucleosides of adenine, in expression or activity of extracellular ectonucleotidases and in activation of P2X and P2Y receptors. Among the best known of these diseases are atherosclerosis, hypertension, cancer, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). The currently available treatments present limited effectiveness and are mostly palliative. This review aims to present the role of purinergic signaling highlighting the ectonucleotidases E-NTPDase, E-NPP, E-5'-nucleotidase, and adenosine deaminase in noncommunicable, neurological, and degenerative diseases associated with the cardiovascular and central nervous systems and cancer. In conclusion, changes in the activity of ectonucleotidases were verified in all reviewed diseases. Although the role of ectonucleotidases still remains to be further investigated, evidences reviewed here can contribute to a better understanding of the molecular mechanisms of highly complex diseases, which majorly impact on patients' quality of life.


Assuntos
Doenças Cardiovasculares/enzimologia , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Nucleotidases/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Humanos , Doenças não Transmissíveis , Qualidade de Vida , Transdução de Sinais
4.
Biomed Pharmacother ; 103: 1701-1707, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29864960

RESUMO

This study evaluated the influence of colistin sulphate on cholinergic, monoaminergic, purinergic and oxidative stress biomarkers in the brain of male rats. Rats were randomized into four (4) groups (A-D) of five rats each. Group A rats received the vehicle of administration for 7 days. Rats in groups B, C and D received 5-, 7.5- and 15-mg/kg body weight colistin sulphate intravenously for 7 days. Colistin sulphate administration significantly raised the narrow beam, landing foot spread distance and gait scores. Administration of colistin sulphate dose dependently increased the activities of acetylcholinesterase, butyrylcholinesterase, monoamine oxidases A and B, ecto-nucleoside triphosphate diphosphohydrolase and ecto-5' nucleotidase. Furthermore, the activities of superoxide dismutase, catalase and glutathione S-tranferase activities in the brain of rats treated with colistin sulphate decreased significantly. Similarly, colistin sulphate lowered the level of reduced glutathione. Caspase-3, malondialdehyde and fragmented DNA in the brain of rats were significantly raised. Colistin sulphate induced gross nuclear condensation and depletion, in addition to cytoplasmic degenerative changes and dilated blood vessels in the brain of rats. Available data from this study show that alterations in the cholinergic, monoaminergic, purinergic and oxidative stress biomarkers are associated with colistin sulphate-mediated neurotoxicity.


Assuntos
Acetilcolina/metabolismo , Monoaminas Biogênicas/metabolismo , Biomarcadores/metabolismo , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Estresse Oxidativo , Receptores Purinérgicos/metabolismo , Acetilcolinesterase/metabolismo , Administração Intravenosa , Animais , Antioxidantes/metabolismo , Comportamento Animal , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Colistina/administração & dosagem , Marcha , Masculino , Nucleotidases/metabolismo , Ratos Wistar
5.
Int J Biol Macromol ; 114: 776-787, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29580999

RESUMO

The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.


Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/enzimologia , Nucleotidases/química , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Nucleotidases/síntese química , Nucleotidases/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/fisiologia , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Especificidade por Substrato , Temperatura Ambiente , Zinco/química
6.
Arch Oral Biol ; 85: 201-206, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29127888

RESUMO

BACKGROUND: Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. OBJECTIVE: To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. METHODS: Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. RESULTS: Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. CONCLUSION: The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes.


Assuntos
Vesículas Extracelulares/enzimologia , Histamina/farmacologia , Nucleotidases/metabolismo , Glândula Submandibular/metabolismo , Animais , Técnicas In Vitro , Indóis/farmacologia , Masculino , Microscopia Eletrônica de Transmissão , Piperazinas/farmacologia , Ratos , Ratos Wistar
7.
Microb Pathog ; 115: 64-67, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29253595

RESUMO

It is recognized that the purinergic system, through the activities of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-nucleotidase), and ecto-adenosine deaminase (E-ADA), is involved in the regulation and modulation of the physiological and pathological events linked to hemostasis. This occurs due to the role of adenosine diphosphate (ADP) in the activation and recruitment of platelets, and the role of adenosine (Ado) in the inhibition of platelet activation. Thus, here we aimed to evaluate whether Aeromonas caviae infection impairs the ecto-enzymes of the purinergic system in fish thrombocytes and the involvement of this system in the hemorrhagic septicemia. The total number of fish thrombocytes decreased in infected animals compared to uninfected animals. Regarding the ecto-enzymes of the purinergic system, the E-NTPDase and E-5'-nucleotidase activities increased in infected animals compared to uninfected animals, while the E-ADA activity decreased. These findings show that adenine nucleotide hydrolysis is modified in the thrombocytes of fish experimentally infected with A. caviae, which impairs the coagulation process due the excessive hydrolysis of ADP, a molecule linked with activation and recruitment of thrombocytes at the site of vascular injury, and augmentation on Ado levels, a molecule linked with inhibitory effects on platelet activation and aggregation. In summary, the purinergic system might contribute to the occurrence of hemorrhagic frames in fish infected with A. caviae.


Assuntos
Aeromonas caviae/patogenicidade , Plaquetas/metabolismo , Ativação Enzimática , Infecções por Bactérias Gram-Negativas/veterinária , Nucleotidases/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina/fisiologia , Adenosina Desaminase , Animais , Brasil , Peixes-Gato/microbiologia , Doenças dos Peixes/microbiologia , Peixes , Hidrólise , Pirofosfatases
8.
Plant J ; 92(6): 1170-1181, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29078035

RESUMO

By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re-established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage-binding protein 2 (DDB2) and the ROS1-mediated active DNA demethylation pathway in Arabidopsis thaliana. We show that DDB2 forms a complex with ROS1 and AGO4 and that they act at the ROS1 locus to modulate levels of DNA methylation and therefore ROS1 expression. We found that DDB2 represses enzymatic activity of ROS1. DNA demethylation intermediates generated by ROS1 are processed by the DNA 3'-phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE1L, and we also show that DDB2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB2 acts as a critical regulator of ROS1-mediated active DNA demethylation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Dano ao DNA , Desmetilação do DNA , Metilação de DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/genética , Nucleotidases/genética , Nucleotidases/metabolismo
9.
Purinergic Signal ; 13(4): 489-496, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28815408

RESUMO

The aim of this study was to verify the effect of diphenyl diselenide (PhSe)2 on hepatic nucleotidases and on the concentration of purines in mice infected by Toxoplasma gondii. The animals were divided into four groups: Group A (uninfected), Group B (uninfected and treated with (PhSe)2), Group C (infected), and Group D (infected and treated with (PhSe)2). The inoculation (groups C and D) was performed with 50 cysts of T. gondii (ME-49 strain). Mice from groups B and D were treated with 5 µmol kg-1 of (PhSe)2. Liver tissue from infected mice showed less severe inflammation, elevated ATP/ADO ratio, elevated NTPDase, 5'nucleotidase, and ADA activities compared to the uninfected group (Group A; P < 0.05). However, infected and treated mice showed decreased ATP levels and elevated ADO levels, as well as higher NTPDase and 5'nucleotidase activities and decreased ADA activity in the hepatic tissue compared to the infected group (P < 0.05). Moreover, the (PhSe)2 treatment of infected mice reduced the hepatic inflammation and showed an immunomodulatory effect on ectonucleotidases of hepatic lymphocytes, which it returned to basal levels. Therefore, chronic infection by T. gondii induces hepatic inflammation in mice, and it is possible that purine levels and nucleotidase activities in hepatic tissue are related to the pathogenesis of the infection in this tissue. The treatment with (PhSe)2 was able to reverse the hepatic inflammation in mice chronically infected, possibly due to the modulation of purinergic enzymes that produce an anti-inflammatory profile through the purinergic system in the liver tissue.


Assuntos
Derivados de Benzeno/farmacologia , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Compostos Organosselênicos/farmacologia , Toxoplasmose/patologia , Animais , Camundongos , Nucleotidases/efeitos dos fármacos , Nucleotidases/metabolismo , Purinas/metabolismo
10.
Infect Immun ; 85(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28717030

RESUMO

Ecto-5'-nucleotidase (CD73) is expressed abundantly on the apical surface of intestinal epithelial cells (IECs) and functions as the terminal enzyme in the generation of extracellular adenosine. Previous work demonstrated that adenosine signaling in IECs results in a number of tissue-protective effects during inflammation; however, a rationale for its apical expression has been lacking. We hypothesized that the highly polarized expression of CD73 is indicative of an important role for extracellular adenosine as a mediator of host-microbe interactions. We show that adenosine harbors bacteriostatic activity against Salmonella enterica serovar Typhimurium that is not shared by the related purine metabolite 5'-AMP, inosine, or hypoxanthine. Analysis of Salmonella colonization in IEC-specific CD73 knockout mice (CD73f/fVillinCre ) revealed a nearly 10-fold increase in colonization compared to that in controls. Despite the increased luminal colonization by Salmonella, CD73f/fVillinCre mice were protected against Salmonella colitis and showed reduced Salmonella burdens in viscera, suggesting that adenosine promotes dissemination. The knockdown of CD73 expression in cultured IECs resulted in dramatic defects in intraepithelial localization and replication as well as defective transepithelial translocation by Salmonella In conclusion, we define a novel antimicrobial activity of adenosine in the gastrointestinal tract and unveil an important role for adenosine as a regulator of host-microbe interactions. These findings have broad implications for the development of new therapeutic agents for infectious disease.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Interações Hospedeiro-Patógeno , Mucosa Intestinal/microbiologia , Salmonella enterica/crescimento & desenvolvimento , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Adenosina/imunologia , Animais , Carga Bacteriana , Linhagem Celular , Células Epiteliais/microbiologia , Inflamação , Camundongos , Camundongos Knockout , Nucleotidases/metabolismo , Salmonella enterica/fisiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologia , Transdução de Sinais
11.
Exp Parasitol ; 181: 7-13, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28710007

RESUMO

Toxoplasma gondii, an intracellular protozoan, may cause chronic infection in the brain tissue of the host inducing a systemic pro-inflammatory profile. Chronic infections can induce numerous physiological changes, such as alterations in the immune and oxidative profiles. Diphenyl diselenide (PhSe)2, an organoselenium compound, has shown antioxidant and immunomodulatory activities in recent studies. So, the aim of this study was to investigate the activity of purinergic enzymes and reactive oxygen species (ROS) in serum and spleen of mice chronically infected by T. gondii, untreated and treated with (PhSe)2. For this experiment, were divided into four groups: Group A (healthy mice), Group B (healthy mice treated with (PhSe)2), Group C (infected mice) and Group D (infected mice treated with (PhSe)2). Group C and group D were infected via oral route with ME49 Toxoplasma gondii strain. Groups B and D were treated subcutaneously with 5 µmol kg-1 of (PhSe)2. Chronic T. gondii infection induced splenomegaly and physiological changes in the spleen and raised histologic inflammatory markers, ROS levels and the activity of purinergic enzymes activity such as NTPDase, 5´nucleotidase and ADA. In serum, the infection increased 5´nucleotidase and ADA activities. (PhSe)2per se has managed to decrease ROS levels and ADA activity and increase NTPDase and 5´nucleotidase in spleen. In infected mice, treatment with (PhSe)2 reversed splenomegaly, reduced histological inflammatory markers, ROS levels and ADA activity in the spleen. Our results prove that chronic toxoplasmosis can induce splenomegaly, heightens ROS levels and purinergic enzyme activity in mice. These results suggest that (PhSe)2 is a potential therapy for the alterations found in the spleen in chronic T. gondii infection.


Assuntos
Derivados de Benzeno/uso terapêutico , Nucleotidases/sangue , Compostos Organosselênicos/uso terapêutico , Baço/patologia , Toxoplasmose Animal/tratamento farmacológico , 5'-Nucleotidase/sangue , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Derivados de Benzeno/farmacologia , Feminino , Inflamação/tratamento farmacológico , Camundongos , Nucleotidases/metabolismo , Compostos Organosselênicos/farmacologia , Espécies Reativas de Oxigênio/sangue , Espécies Reativas de Oxigênio/metabolismo , Baço/efeitos dos fármacos , Baço/enzimologia , Baço/metabolismo , Toxoplasmose Animal/enzimologia , Toxoplasmose Animal/patologia
12.
Exp Parasitol ; 179: 1-6, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28587841

RESUMO

3'-nucleotidase/nuclease (3'NT/NU) is a bi-functional enzyme that is able to hydrolyze 3'-monophosphorylated nucleotides and nucleic acids. This review summarizes the major molecular and biochemical properties of this enzyme in different trypanosomatid species. Sequence analysis of the gene encoding 3'NT/NU in Leishmania and Crithidia species showed that the protein possesses five highly conserved regions that are characteristic of members of the class I nuclease family. 3'NT/NU presents a molecular weight of approximately 40 kDa, which is conserved among the studied species. Throughout the review, we discuss inhibitors and substrate specificity, relating them to the putative structure of the enzyme. Finally, we present the major biological roles performed by 3'NT/NU. The involvement of 3'NT/NU in the purine salvage pathway was confirmed by the increase of activity and expression of the enzyme when the parasites were submitted to purine starvation. The generation of extracellular adenosine is also important to the modulation of the host immune response. Interaction assays involving Leishmania parasites and macrophages indicated that 3'-nucleotidase activity increases the association index between them. Recently, it was shown that 3'NT/NU plays a role in parasite escape from neutrophil extracellular traps, one of the first mechanisms of the host immune system for preventing infection.


Assuntos
Nucleotidases/metabolismo , Trypanosomatina/enzimologia , Interações Hospedeiro-Parasita , Concentração de Íons de Hidrogênio , Macrófagos/parasitologia , Nucleotidases/antagonistas & inibidores , Nucleotidases/química , Nucleotidases/genética , Especificidade por Substrato , Trypanosomatina/genética
13.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 5): 276-280, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28471359

RESUMO

2',3'-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6 Šresolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.


Assuntos
Proteínas de Bactérias/química , Deinococcus/química , Nucleotidases/química , RNA Bacteriano/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Deinococcus/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Nucleotidases/genética , Nucleotidases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Biotechnol Lett ; 39(8): 1211-1217, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28497175

RESUMO

OBJECTIVES: A 2',3'-cyclic phosphodiesterase gene (drCPDase) has been characterized from Deinococcus radiodurans and is involved in the robust resistance of this organism. RESULTS: Cells lacking 2',3'-cyclic phosphodiesterase gene (drCPDase) showed modest growth defects and displayed increased sensitivities to high doses of various DNA-damaging agents including ionizing radiation, mitomycin C, UV and H2O2. The transcriptional level of drCPDase increased after H2O2 treatment. Additional nucleotide monophosphate partially recovered the phenotype of drCPDase knockout cells. Complementation of E. coli with drCPDase resulted in enhanced H2O2 resistance. CONCLUSIONS: The 2',3'-cyclic phosphodiesterase (drCPDase) contributes to the extreme resistance of D. radiodurans and is presumably involved in damaged nucleotide detoxification.


Assuntos
Deinococcus/enzimologia , Nucleotidases/metabolismo , Proteínas Recombinantes/metabolismo , Deinococcus/genética , Escherichia coli/genética , Peróxido de Hidrogênio , Viabilidade Microbiana/genética , Mutação , Nucleotidases/química , Nucleotidases/genética , Estresse Oxidativo/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
15.
Mol Ther ; 25(5): 1209-1221, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28330694

RESUMO

Uveal melanoma (UM) is an intraocular malignant tumor with a high mortality rate. Recent studies have shown the functions of long non-coding RNAs (lncRNAs) in tumorigenesis; thus, targeting tumor-specific lncRNA abnormalities has become an attractive approach for developing therapeutics to treat uveal melanoma. In this study, we identified a novel nuclear CANT1 lncRNA (CASC15-New-Transcript 1) that acts as a necessary UM suppressor. CANT1 significantly reduced tumor metastatic capacity and tumor formation, either in cell culture or in animals harboring tumor xenograft. Intriguingly, XIST lncRNA serves as a potential target of CANT1, and JPX or FTX lncRNA subsequently serves as a contextual hinge to activate a novel CANT1-JPX/FTX-XIST long non-coding (lncing) pathway in UM. Moreover, CANT1 triggers the expression of JPX and FTX by directly binding to their promoters and promoting H3K4 methylation. These observations delineate a novel lncing cascade in which lncRNAs directly build a lncing cascade without coding genes that aims to modulate UM tumorigenesis, thereby specifying a novel "lncing-cascade renewal" anti-tumor therapeutic strategy by correcting aberrant lncing cascade in uveal melanoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/terapia , Nucleotidases/genética , Plasmídeos/metabolismo , RNA Longo não Codificante/genética , Neoplasias Uveais/terapia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Injeções Subcutâneas , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Nucleotidases/metabolismo , Plasmídeos/química , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Res Vet Sci ; 111: 21-25, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28266315

RESUMO

Avian colibacillosis is one of the most common infectious diseases caused partially or entirely by avian pathogenic Escherichia coli (APEC) in birds. In addition to spontaneous infection, APEC can also cause secondary infections that result in greater severity of illness and greater losses to the poultry industry. In order to assess the role of 2', 3'-cyclic phosphodiesterase (cpdB) in APEC on disease physiology and pathogenicity, an avian pathogenic Escherichia coli-34 (APEC-34) cpdB mutant was obtained using the Red system. The cpdB mutant grew at a slower rate than the natural strain APEC-34. Scanning electron microscopy (SEM) indicated that the bacteria of the cpdB mutant were significantly longer than the bacteria observed in the natural strain (P<0.01), and that the width of the cpdB mutant was significantly smaller than its natural counterpart (P<0.01). In order to evaluate the role of cpdB in APEC in the colonization of internal organs (lung, liver and spleen) in poultry, seven-day-old SPF chicks were infected with 109CFU/chick of the cpdB mutant or the natural strain. No colonizations of cpdB mutants were observed in the internal organs 10days following the infection, though numerous natural strains were observed at 20days following infection. Additionally, the relative expression of division protein ftsZ, outer membrane protein A ompA, ferric uptake regulator fur and tryptophanase tnaA genes in the mutant strain were all significantly lower than in the natural strain (P<0.05 or P<0.01). These results suggested that cpdB is involved in the long-term colonization of APEC in the internal organs of the test subjects. The deletion of the cpdB gene also significantly affected the APEC growth and morphology.


Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Nucleotidases/metabolismo , Doenças das Aves Domésticas/microbiologia , Animais , Escherichia coli/patogenicidade , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Nucleotidases/genética , Virulência , Fatores de Virulência/genética
17.
Curr Genet ; 63(2): 229-239, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27387517

RESUMO

The yeast Saccharomyces cerevisiae is capable of responding to various environmental stresses, such as salt stress. Such responses require a complex network and adjustment of the gene expression network. The goal of this study is to further understand the molecular mechanism of salt stress response in yeast, especially the molecular mechanism related to genes BDF1 and HAL2. The Bromodomain Factor 1 (Bdf1p) is a transcriptional regulator, which is part of the basal transcription factor TFIID. Cells lacking Bdf1p are salt sensitive with an abnormal mitochondrial function. We previously reported that the overexpression of HAL2 or deletion of HDA1 lowers the salt sensitivity of bdf1Δ. To better understand the mechanism behind the HAL2-related response to salt stress, we compared three global transcriptional profiles (bdf1Δ vs WT, bdf1Δ + HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs bdf1Δ) in response to salt stress using DNA microarrays. Our results reveal that genes for iron acquisition and cellular and mitochondrial remodeling are induced by HAL2. Overexpression of HAL2 decreases the concentration of nitric oxide. Mitochondrial iron-sulfur cluster (ISC) assembly also decreases in bdf1Δ + HAL2. These changes are similar to the changes of transcriptional profiles induced by iron starvation. Taken together, our data suggest that mitochondrial functions and iron homeostasis play an important role in bdf1Δ-induced salt sensitivity and salt stress response in yeast.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Ferro/metabolismo , Nucleotidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Tolerância ao Sal/genética , Fatores de Transcrição/genética , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Immunoblotting , Mutação , Nucleotidases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Fatores de Transcrição/metabolismo
18.
BMC Microbiol ; 16(1): 249, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27784292

RESUMO

BACKGROUND: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified. RESULTS: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not. CONCLUSIONS: Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.


Assuntos
5'-Nucleotidase/metabolismo , Bacillus subtilis/enzimologia , 5'-Nucleotidase/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus subtilis/genética , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Hidrolases/genética , Hidrolases/metabolismo , Fosfatos de Inositol/metabolismo , Nucleotidases/metabolismo , Estresse Oxidativo/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Homologia de Sequência , Células-Tronco , Especificidade por Substrato
19.
Trends Biochem Sci ; 41(12): 1050-1060, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27658684

RESUMO

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways.


Assuntos
Adenosina Trifosfatases/química , Evolução Molecular , GTP Fosfo-Hidrolases/química , Fatores de Troca do Nucleotídeo Guanina/química , Nucleotidases/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/classificação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , GTP Fosfo-Hidrolases/classificação , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Nucleotidases/classificação , Nucleotidases/genética , Nucleotidases/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Exp Parasitol ; 169: 111-8, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27531705

RESUMO

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.


Assuntos
Guanosina Difosfato/farmacologia , Guanosina Monofosfato/farmacologia , Guanosina Trifosfato/farmacologia , Leishmania mexicana/enzimologia , Nucleotidases/antagonistas & inibidores , Animais , Cinética , Leishmania mexicana/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Nucleotidases/metabolismo , Células RAW 264.7
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