Laboratorio Of Biologia Genomica E Molecular

Porto Alegre, Brazil

Laboratorio Of Biologia Genomica E Molecular

Porto Alegre, Brazil
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Rico E.P.,Institute Ciencias Basicas da Sau | Rico E.P.,Instituto Nacional em Excitotoxicidade e Neuroprotecao INCT EN | de Oliveira D.L.,Institute Ciencias Basicas da Sau | de Oliveira D.L.,Instituto Nacional em Excitotoxicidade e Neuroprotecao INCT EN | And 13 more authors.
Brain Research Bulletin | Year: 2010

High-affinity excitatory amino acid transporters (EAATs) regulate extracellular glutamate levels. Zebrafish (Danio rerio) provides an excellent model to study the function of different neurotransmitter systems. Although the identification of the EAAT family is well established in the mammalian central nervous system (CNS), EAAT-related genes and their expression profile in zebrafish have not yet been reported. Here we identify and describe the expression profile of EAATs-related genes and functional properties of glutamate uptake in three major brain structures from zebrafish (telencephalon, optic tectum and cerebellum). Searches on zebrafish genome databases and a phylogenetic analysis confirmed the presence of several EAAT-related genes (EAAT2, EAAT3, three EAAT1 paralogs and two EAAT5 sequences). All sequences identified were expressed in the structures analyzed. EAAT2 and EAAT3 were the most prominent glutamate transporters expressed in all brain areas. A uniform expression was observed for EAAT1A, whereas higher EAAT1B transcript levels were detected in telencephalon. Lower amounts of EAAT1C transcripts were observed in cerebellum when compared to other structures. No EAAT4-related sequence was found in the zebrafish genome. The EAAT5A expression was similar to EAAT5B in the telencephalon, while EAAT5B was less expressed than EAAT5A in optic tectum and cerebellum. Moreover, the glutamate uptake was significantly higher in optic tectum, which indicates functional differences within zebrafish brain structures. Altogether, the study of glutamate uptake in zebrafish could be important to evaluate the modulation of glutamatergic signaling through pharmacological and toxicological studies. © 2009 Elsevier Inc. All rights reserved.


Rico E.P.,Federal University of Rio Grande do Sul | Rico E.P.,Instituto Nacional Of Ciencia E Tecnologia Em Excitotoxicidade E Neuroprotecao | Rosemberg D.B.,Federal University of Rio Grande do Sul | Rosemberg D.B.,Instituto Nacional Of Ciencia E Tecnologia Em Excitotoxicidade E Neuroprotecao | And 10 more authors.
NeuroToxicology | Year: 2011

Ethanol is a widely consumed drug that acts on the central nervous system (CNS), modifying several signal transduction pathways activated by hormones and neurotransmitters. The zebrafish is an experimental model for the study of human diseases and the use of this species in biochemical and behavioral studies on alcoholism and alcohol-dependence has increased recently. However, there are no data concerning the effects of chronic ethanol exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. Purinergic signaling is controlled by a group of enzymes named ectonucleotidases, which include NTPDases and ecto-5'-nucleotidase already characterized in zebrafish brain. The aim of this study was to evaluate nucleotide hydrolysis by NTPDases and ecto-5'-nucleotidase after long-term ethanol exposure. Additionally, the gene expression patterns of NTPDases1-3 and 5'-nucleotidase were determined. Animals were exposed to 0.5% ethanol for 7, 14, and 28 days. There were no significant changes in ATP and GTP hydrolysis after all treatments. However, a decrease in ADP (46% and 34%) and GDP (48% and 36%) hydrolysis was verified after 7 and 14 days, respectively. After 7 and 14 days of ethanol exposure, a significant decrease in AMP hydrolysis (48% and 36%) was also observed, whereas GMP hydrolysis was inhibited only after 7 days (46%). NTPDase2_mv and NTPDase3 mRNA transcript levels decreased after 7 and 14 days, respectively. In contrast, ethanol increased NTPDase1, NTPDase2_mq, and NTPDase3 transcript levels after 28 days of exposure. NTPDase2_mg and 5'-nucleotidase gene expression was not altered. Therefore, the ectonucleotidase pathway may be a target of chronic ethanol toxicity and the regulation of purinergic system could play a key role in the neurochemical mechanisms underlying the effects of ethanol on the CNS. © 2011 Elsevier Inc.


Altenhofen S.,Laboratorio Of Neuroquimica E Psicofarmacologia | Nabinger D.D.,Laboratorio Of Neuroquimica E Psicofarmacologia | Pereira T.C.B.,Laboratorio Of Biologia Genomica E Molecular | Leite C.E.,Institute Toxicologia e Farmacologia | And 3 more authors.
Molecular Neurobiology | Year: 2017

ATP and adenosine, the main signaling molecules of purinergic system, are involved in toxicological effects induced by metals. The manganese (Mn) exposure induces several cellular changes, which could interfere with signaling pathways, such as the purinergic system. In this study, we evaluated the effects of exposure to manganese(II) chloride (MnCl2) during 96 h on nucleoside triphosphate diphosphohydrolase (NTPDase), ecto-5′-nucleotidase, and adenosine deaminase (ADA) activities, followed by analyzing the gene expression patterns of NTPDases (entpd1, entpd2a.1, entpd2a.2, entpd2-like, entpd3) and ADA (ADA1, ADA2.1, ADA2.2, ADAasi, ADAL) families in zebrafish brain. In addition, the brain metabolism of nucleotides and nucleosides was evaluated after MnCl2 exposure. The results showed that MnCl2 exposure during 96 h inhibited the NTPDase (1.0 and 1.5 mM) and ecto-ADA (0.5, 1.0, and 1.5 mM) activities, further decreasing ADA2.1 expression at all MnCl2 concentrations analyzed. Purine metabolism was also altered by the action of MnCl2. An increased amount of ADP appeared at all MnCl2 concentrations analyzed; however, AMP and adenosine levels are decreased at the concentrations of 1.0 and 1.5 mM MnCl2, whereas decreased inosine (INO) levels were observed at all concentrations tested. The findings of this study demonstrated that MnCl2 may inhibit NTPDase and ecto-ADA activities, consequently modulating nucleotide and nucleoside levels, which may contribute for the toxicological effects induced by this metal. © 2017 Springer Science+Business Media New York


Vieira P.B.,Federal University of Rio Grande do Sul | Silva N.L.F.,Federal University of Rio Grande do Sul | Kist L.W.,Laboratorio Of Biologia Genomica E Molecular | Oliveira G.M.T.,Laboratorio Of Biologia Genomica E Molecular | And 4 more authors.
Memorias do Instituto Oswaldo Cruz | Year: 2015

Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5’-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalis isolates from female and male patients were evaluated. Iron from different sources sustained T. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5’-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron. © 2015, Fundacao Oswaldo Cruz. All rights reserved.


Menezes F.P.,Laboratorio Of Neuroquimica E Psicofarmacologia | Kist L.W.,Laboratorio Of Biologia Genomica E Molecular | Bogo M.R.,Laboratorio Of Biologia Genomica E Molecular | Bonan C.D.,Laboratorio Of Neuroquimica E Psicofarmacologia | And 3 more authors.
Zebrafish | Year: 2015

Imbalances in glutamatergic signaling have been proposed as the cause of several neurological disturbances. The use of MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, to mimic features of these neurological disorders is effective both in mammals and in fish. However, the variability of the subunits comprising the NMDA receptor during development alters the pharmacokinetic properties of the receptor and leads to different responses to this drug. Here, we evaluated the locomotor response of zebrafish to MK-801 (1, 5, and 20 μM) through the development (30 days postfertilization [dpf] to 2 years postfertilization [ypf]). The NMDA receptor subunit gene expression was also analyzed through the development (7 dpf to 2 ypf). Zebrafish displayed an age-related response to MK-801 with a higher response at 60 and 120 dpf. The magnitude of hyperlocomotion promoted by MK-801 seems to be less powerful for zebrafish in relation to rodents. The verification of expression levels in zebrafish NMDA receptor subunits shows that NR1.1 had a slight reduction throughout the development, while the NR2 subunits, especially NR2A.2 and NR2C.1, vary their expression levels according to the stage of development. The time-specific locomotor response to MK-801 through the development could be a consequence of differential NMDA receptor subunit expression. This result of developmental response to MK-801 is a crucial component in the consolidation of zebrafish as a suitable model to study glutamatergic neurotransmission in early phases. © 2015, Mary Ann Liebert, Inc.


Capiotti K.M.,Grande Rio University | Siebel A.M.,Instituto Nacional Of Ciencia E Tecnologia Translacional Em Medicina Inct Tm | Siebel A.M.,Chapecó Region Community University | Kist L.W.,Laboratorio Of Biologia Genomica E Molecular | And 7 more authors.
Purinergic Signalling | Year: 2016

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 (adora1, adora2aa, adora2ab, and adora2b) 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. © 2016 Springer Science+Business Media Dordrecht


Frasson A.P.,Laboratorio Of Pesquisa Em Parasitologia Tiana | Charao M.F.,Laboratorio Of Toxicologia | Rosemberg D.B.,Federal University of Rio Grande do Sul | Garcia S.C.,Laboratorio Of Toxicologia | And 5 more authors.
Memorias do Instituto Oswaldo Cruz | Year: 2012

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterized in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.


Rosemberg D.B.,Institute Ciencias Basicas da Sau | Rosemberg D.B.,Instituto Nacional em Excitotoxicidade e Neuroprotecao INCT EN | Rico E.P.,Institute Ciencias Basicas da Sau | Rico E.P.,Instituto Nacional em Excitotoxicidade e Neuroprotecao INCT EN | And 10 more authors.
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology | Year: 2010

The nucleoside triphosphate diphosphohydrolase (NTPDase) family cleaves tri- and diphosphonucleosides to monophosphonucleosides and is responsible for terminating purinergic transmission. Since the NTPDase family in zebrafish is poorly understood, here we evaluated the nucleotide hydrolysis in three tissues of adult zebrafish (brain, liver, and heart), confirmed the presence of distinct NTPDase members by a phylogenetic analysis and verified their relative gene expression profiles in the respective tissues. A different profile of ATP and ADP hydrolysis in the brain, liver, and heart as a function of time and protein concentration was observed. Sodium azide (20 mM), ARL 67156 (300 μM) and Suramin (300 μM) differently altered the nucleotide hydrolysis in zebrafish tissues, suggesting the contribution of distinct NTPDase activities. Homology-based searches identified the presence of NTPDase1-6 and NTPDase8 orthologs and the phylogeny also grouped three NTPDase2 and two NTPDase5 paralogs. The deduced amino acid sequences share the apyrase conserved regions, conserved cysteine residues, putative N-glycosylation, phosphorylation, N-acetylation sites, and different numbers of transmembrane domains. RT-PCR experiments revealed the existence of a distinct relative entpd1-6 and entpd8 expression profile in brain, liver, and heart. Taken together, these results indicate that several NTPDase members might contribute to a tight regulation of nucleotide hydrolysis in zebrafish tissues. © 2009 Elsevier Inc. All rights reserved.


Siebel A.M.,Laboratorio Of Neuroquimica E Psicofarmacologia | Menezes F.P.,Laboratorio Of Neuroquimica E Psicofarmacologia | Capiotti K.M.,Laboratorio Of Neuroquimica E Psicofarmacologia | Kist L.W.,Laboratorio Of Biologia Genomica E Molecular | And 5 more authors.
Zebrafish | Year: 2015

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5′nucleotidase inhibitor adenosine 5′-(α,β-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5′-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish. © 2015, Mary Ann Liebert, Inc.

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