2C, lane 4) a partial recovery of TvCP39 amount was observed

2C, lane 4) a partial recovery of TvCP39 amount was observed. mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization. Introduction Trichomonosis is the most common non-viral sexually transmitted contamination (STI) caused by cysteine proteinases (CPs) play important roles in trichomonad pathogenesis such as cytoadherence, immune evasion, haemolysis, and cytotoxicity [7]C[12]. The synthesis and proteolytic activity of certain CPs are regulated by environmental factors such as iron, pH, oxidation-reduction capacity, temperature, and polyamines [9],[11],[13]C[15]. The 39 kDa CP (TvCP39), which was found in vaginal washes from patients with trichomonosis and it is localized in the parasite surface, is usually involved in cytotoxicity to HeLa, DU145 and vaginal epithelial cells (VECs). Interestingly, this CP is usually and secreted by treated with 20 mM DAB resulted in growth arrest. Additionally, the amount of adhesins involved in trichomonal adherence did not change in DAB-treated parasites; however, an increase in adherence was observed [16]. Interestingly, the addition of 40 mM putrescine to DAB-treated trichomonads was used to rescue growth arrest, and reduced the elevated levels of adherence [16]. Since in TvCP65 is usually involved in trichomonal cytotoxicity and the expression, protein amount, and proteolytic activity of this CP were reduced in DAB-treated parasites, we suggested that probably exist a relationship between the parasite virulence and polyamines concentration in expression, mRNA stability and proteolytic activity, but also the TvCP39 cellular localization. Materials and Methods 1. culture and inhibition/restoration of putrescine metabolism Late-logarithmic-phase trophozoites of isolate CNCD147 grown for 24 h in Diamond’s trypticase-yeast extract-maltose (TYM) medium pH 6.2 with 10% heat-inactivated horse serum (Gibco) (normal media) at 37C were used for all assays. The putrescine metabolism inhibition was performed as previously reported [14],[22]. Parasite viability after these treatments was checked by the trypan blue (Sigma) exclusion method [23]. 2. RNA extraction and cDNA synthesis Total RNA from 2107 parasites grown in the absence or presence of 20 mM DAB in TYM medium for 24 h, and DAB-treated parasites transferred into 40 mM exogenous putrescine medium for 30 min at 37C and into TYM medium (as a control). The RNA was extracted using TRizol reagent (Invitrogen), according to the manufacturer’s protocol. Purified RNA was digested with DNase I (Invitrogen) to discard the DNA contaminant, according to the manufacturer’s protocol. RNA concentration and purity were determined by measuring absorbance using NanoDrop 2000 (Thermo Scientific); all 260/280 ratios were between 1.8 and 2.1. Then, 1 g of total RNA was reverse-transcribed using the Superscript II Reverse Transcriptase Kit (Invitrogen), according to the manufacturer’s protocol using the oligo-dT (dT18) (10 pmol/l) primer. 3. Analysis of expression by semi-quantitative and quantitative RT-PCR To validate the expression of in different putrescine conditions, RT-PCR analysis were performed using 50 ng cDNA from parasites grown in the absence or presence of 20 SYM2206 mM DAB, or DAB-treated parasites transferred into 40 mM exogenous putrescine medium, 10 pmol of each primer pair and 0.25 U of Taq SYM2206 DNA polymerase (Invitrogen). PCR was carried out in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems Inc., Foster City, CA, USA). Specific primer pairs were designed using Primer3 software version 3.0 (www.primer3.sourceforge.net). We used the following primer pairs to amplify: 110 bp of the gene (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_001316379″,”term_id”:”123457372″,”term_text”:”XM_001316379″XM_001316379), sense (CP39-FRT) 5 3 and antisense (CP39-RRT) 5 3; and 112 bp of the gene as reported [24]. The amplified products were analyzed on 2% agarose gels and visualized by ethidium bromide staining. Gene expression densitometry analyses were performed using the Quantity One Software (BioRad). Data from densitometry quantification of the housekeeping gene (expression was calculated after the threshold cycle ((in different Ace2 putrescine conditions was expressed as normalized Ct values. All reactions including no-template and RT minus controls for each mRNA SYM2206 were run in triplicate. All experimental data were expressed as means standard deviation (SD) from three individual biological experiments. The significance of the difference between means was determined by ANOVA with Prisma Firewall 1.53 software. The level of significance was also determined by the.