Bogo, Mauricio Reis

Charão, Mariele Feiffer

De Carli, Geraldo Attilio

Frasson, Amanda Piccoli

Garcia, Solange Cristina

Rosemberg, Denis Broock

Tasca, Tiana

English

Lume 5.8

caderno d•farmácla ISSN 0102-6593 Órgão Oficial da Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul volume 26, Suplemento, 201 O INVESTIGAÇÃO DE ALVOS TERAPÊUTICOS E BIOMARCADORES =f + O) O 5 lnvestigation of therapeutic targets and biomarkers 156--------------~--~=-=-~-----------------------------------------------BOVINE SERUM LIMITATION INCREASES THE ACTIVITY ANO EXPRESSION OF ENZYMES INVOLVED ON ATP, ADPAND AMP EXTRACELLULAR HYDROLYSIS IN TR/CHOMONAS VAGINALIS Frasson A.P. 1·; Rosemberg D.B. 2 ; Charão M.F. 3 ; De Carli G.A.4 ; Bogo M.R. 2 ; Garcia S.C. 3 ; Tasca T1. 1Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, UFRGS; 2 Laboratório de Biologia Genômica e Molecular, PUCRS; 3Laboratório de Toxicologia, Faculdade de Farmácia, UFRGS; 41nstituto de Geriatria e Gerontologia, PUCRS *Mestrando - Início: 2009/1 lntroduction: The flagellated protozoan Trichomonas vaginalis causes trichomonosis, the most common non-viral STD in the world 1. The infection causes serious impact on public health; therefore, it is important to investigate the biochemical aspects of the parasite. Extracellular nucleotides are released by cells under stress, anoxia or injuri and they can be inactivated by hydrolysis via ectonucleotidases. The members of E-NTPDase family hydrolyze nucleoside di- and tri-phosphates and the ecto-5'-nucleotidase is able to hydrolyze nucleoside monophosphate, producing adenosine3. lmportant, T. vaginalis lacks the ability to synthesize purines and pyrimidines de novo, and the enzymes act on the salvage pathways generating the nucleosides4 . Objective: The aims of this study were to investigate the metabolism of ATP in trophozoites of T. vaginalis by enzymatic activities determination and gene expression analysis, under a condition of bovine serum limitation. Materiais and Methods: For the assays, the trophozoites from ATCC 30236 and LACH 1 isolates were grown in TYM medium supplemented with 1.0% inactivated bovine serum, while contrai cultures were maintained with 10% serum. lhe enzymatic tests were performed in the reaction mixture containing 50mM Tris buffer, 5.0mM CaCI2 or MgCI2, trophozoites and substrate (1.0 mM ATP, 1.0mM ADP or 3.0mM AMP), at 3rC. The specific activity was determined by colorimetric method and expressed as nmol Pi released/min/mg protein. lhe gene expression patterns were carried out by a RT -PCR assay using specific primers for NTPDase A and B; genes responsible for AMP hydrolysis were searched by BLAST function in GenBank Database and primers were designed for SurE (183), SurE(257), SurE(258) and 5'(3') enzymes. Extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. Results: The bovine serum limitation causes an increase on the hydrolysis of the nucleotides tested. In ATCC 30236, there was an increase of 154.4%, 176.1% and 302.5% on the hydrolysis of ATP, ADP and AMP, respectively, when compared with contrai cultures; in the clinicai isolate, LACH1, the increase was to 14.4%, 22.5% and 162.5%. Transcript leveis of NlPDase A were significantly increased in both isolates, while NTPDase B and SurE(183) mRNA transcript leveis increased only on the LACH1 isolate. Extracellular AlP metabolism is increased in limiting serum, leading to higher ATP hydrolysis and formation of ADP and AMP. Conclusions: Our data suggest that under serum limitation condition these T. vaginalis enzymes play an important role in the provision of adenosine for parasite growth. Moreover, the enzymes may contribute to escape mechanisms ofthe parasite by breaking down ATP. References: 1 Weinstock H et a/. J Persp Sex Reprod. Health 36: 6-1 O, 2004 2 Robson et ai. Purinergic Signal 2: 409-430, 2006 3 Zimmermann H. Drug Develop Res 52: 44-56, 2001 4 Tasca Teta/. Parasitei Res 93: 300-303, 2004 Acknowledgements: A.P.F. is recipient of fellowship from CNPq. This study received financiai support from CNPq (T.T., grant #477348/2008-4). 

Bovine serum limitation increases the activity and expression of enzymes involved on atp, adp and amp extracellular hydrolysis in trichomonas vaginalis

https://lume.ufrgs.br/bitstream/10183/267792/1/000770305.pdf

Bovine serum limitation increases the activity and expression of enzymes involved on atp, adp and amp extracellular hydrolysis in trichomonas vaginalis

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