| Background and objective Trichomonas vaginalis is an unusual eukaryote and has been considered to be one of the earliest branching organisms[1]. It lacks conventional mitochondria but possesses organelles termed hydrogenosomes that share common ancestry with mitochondria[2,3] and appear to be adaptations for the parasite's existence in an environment containing only low oxygen concentrations. Trichomonads are fundamentally fermentative organisms though the cells are exposed to oxygen in the natural environment[4]. Despite a report of the beneficial effect of low oxygen concentrations (growth being significantly enhanced)[5], it is generally considered that oxygen providesproblems rather than benefits. Some of the parasite's enzymes are inactivated by oxygen itself, notably key proteins of the hydrogenosomes, and various metabolites likely to arise from the metabolism of oxygen (such as hydrogen peroxide and hydroxyl free radical) are generally harmful to cells. It was believed that the organism relies heavily upon cytosolic NADH oxidase (reducing oxygen to water) and NADPH oxidase (reducing oxygen to hydrogen peroxide) to prevent permeation of oxygen into the hydrogenosomes[6]. However, the generation of hydrogen peroxide by NADPH oxidase, and also superoxide dismutase, poses the question as to how this and other reactive oxygen species (ROS) are removed as T.vaginalis lacks catalase and glutathione-dependent peroxidase activities[7].A superfamily of peroxiredoxins (Prx) has been identified in a wide variety of organisms ranging from prokaryotes to mammals[8-13] recent years which do not use glutathione as the reductant but instead are dependent on reduction by a small protein known as thioredoxin (Trx), which itself is reduced by thioredoxin reductase (TrxR)[ 14-16]. It has been shown that peroxiredoxins reduce hydrogen peroxide and alkyl hydroperoxides[17-19] and therefore constitute a major cellular protection system against the devastating consequences of oxidative damage. Eukaryotic 2-Cys peroxiredoxins (2-Cys Prxs) not only act as antioxidants, but also appear to regulate hydrogen peroxide-mediated signal transduction[20-21] invovled in cell proliferation, differentiation and apoptosis[22-26] . Peroxiredoxin-linked detoxification may occur in all eukaryotes but has been perceived to be of special relevance to someparasites, including helminths, trypanosomatids and the malaria parasite Plasmodium falciparum. Such metabolism may be of particular importance to amitochondriate eukaryotes, such as the protozoa Entamoeba and Giardia as well as Trichomonas, as they all lack glutathione, but the way in which the enzyme functions has not yet been addressed. In this study, we constructed cDNA library of T. vaginalis, screened the genes of our interest and reported the initial characterization of the T. vaginalis Prx, a novel member of the typical 2-Cys subfamily, and peroxiredoxin-linked functions involved not only in the reduction of alkyl hydroperoxide and hydrogen peroxide as an antioxidant, but also in regulating hydrogen peroxide-mediated signal transduction, which may lay strong foundations for demonstrating the mechanism of aging in organisms of protozoan.Materials and methods (1) Total RNA was isolated from T. vaginalis cells. The concentration of isolated total RNA was determined by a spectrophotometer and the integrity of RNA was analyzed on 1% FA gel.(2) Total RNA sample was reversely transcribed with PowerScript reverse transcriptase. ds cDNA was synthesized by LD_PCR and then digested with enzyme. After purification, the digested fragments were ligated unidirectionally into the Sfi I sites of the XTripIEx2 vector, packaged in vitro, and amplified. The titer and percentage of recombinant clones of the amplified library were determined through blue/white screening.(3) Three hundred cDNA clones were selected from the library at random and converted to pTripIEx2 plasmids. E. coli BM25.8was used as host bacteria for amplification of plasmid DNA cultured in LB...
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