Efficiency of Galanti and Guisti Method of ADA Estimation in Comparison with the Gold Standard

BACKGROUND: Adenosine Deaminase, the key enzyme of purine metabolism catalyzing the irreversible hydrolytic deamination of adenosine to inosine is implicated in a varied spectrum of human diseases ranging from SCID to TB and pneumonia. Estimation of ADA offers an easy, relatively affordable and reliable diagnostic alternative and/ or adjunct (specially in a TB endemic nation) which emphasizes the necessity of a feasible and implementable alternative method to the Diazyme method of ADA estimation requiring high end autoanalyzer and infrastructural setup.METHODS: Sixty body fluids samples (irrespective of gender, age, diagnosis or sample type) received by the Clinical Biochemistry Laboratory, Kasturba Medical College, Manipal for fluid ADA estimation by the Diazyme assay method (cobas 6000) was simultaneously processed by the Galanti and Guisti manual method to estimate the comparability and the aggregability of results obtained by the two analytical techniques.RESULTS: The Galanti and Guisti manual method of ADA estimation showed aggregability with the Diazyme autoanalyzer method for 90% of the assayed study samples with the manual method uniformly showing higher values when compared to the analyzer method. A correction factor of 2.44 was arrived at which could effectively achieve comparability between the two assay methods.CONCLUSION: The Galanti and Guisti manual method of ADA estimation might be a feasible, rapid, reliable and costeffective method for estimation of fluid ADA when compared to the cost and infrastructure intensive autoanalyzer.

RNA editing in mesothelioma: a look forward

RNA editing is a post-transcriptional process increasing transcript diversity, thereby regulating different biological processes. We recently observed that mutations resulting from RNA editing due to hydrolytic deamination of adenosine increase during the development of mesothelioma, a rare cancer linked to chronic exposure to asbestos. This review gathers information from the published literature and public data mining to explore several aspects of RNA editing and their possible implications for cancer growth and therapy. We address possible links between RNA editing and particular types of mesothelioma genetic and epigenetic alterations and discuss the relevance of an edited substrate in the context of current chemotherapy or immunotherapy.

Artificial RNA Editing with ADAR for Gene Therapy

Editing mutated genes is a potential way for the treatment of genetic diseases. G-to-A mutations are common in mammals and can be treated by adenosine-to-inosine (A-to-I) editing, a type of substitutional RNA editing. The molecular mechanism of A-to-I editing involves the hydrolytic deamination of adenosine to an inosine base; this reaction is mediated by RNA-specific deaminases, adenosine deaminases acting on RNA (ADARs), family protein. Here, we review recent findings regarding the application of ADARs to restoring the genetic code along with different approaches involved in the process of artificial RNA editing by ADAR. We have also addressed comparative studies of various isoforms of ADARs. Therefore, we will try to provide a detailed overview of the artificial RNA editing and the role of ADAR with a focus on the enzymatic site directed A-to-I editing.

Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in Trypanosoma brucei: Intracellular Localization and Properties of the Enzyme

ABSTRACT Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2′-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5′-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.

Theoretical study of mechanisms for the hydrolytic deamination of cytosine via steered molecular dynamic simulations

Gibbs free energy profiles of the cytosine deamination assisted by a water molecule in a discrete aqueous medium were obtained by the application of Steered Molecular Dynamic (SMD) simulations.