Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

“…The recent completion of the E. histolytica genome sequence generated a large raw data set that can be used to gain insights into the molecular biology of this organism. The present work and the accompanying paper by López-Camarillo et al (2008) are the first reports about RNA helicases in this protozoan parasite. The EhDEAD and EhDExH-box families of E. histolytica are similar in size to RNA helicases families described in other eukaryotic organisms (de la Cruz et al, 1999;Boudet et al, 2001;Abdelhaleem et al, 2003), including the protozoan parasites L. major (Ivens et al, 2005) and P. falciparum (Tuteja and Pradhan, 2006).…”

The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

“…No signal was detected using pre-immune serum (lanes 3 and 4). The use of antibodies against EhPAP that has been immunodetected in both NE and CE [36], confirmed protein integrity (lanes 5 and 6), while the use of antibodies against the nuclear EhPC4 [42] validated cell fractionation of both extracts (lanes 7 and 8). Intriguingly, anti-EhCFIm25 antibodies revealed a 55 kDa band whereas the predicted molecular weight for the polypeptides referred to as C4M2T1, Q7YZC1 and B1N3I5 is 30.4, 28.1 and 15.2 kDa, respectively.…”

“…As controls, we used anti-EhPC4 antibodies that specifically identified the putative E. histolytica transcription and polyadenylation factor EhPC4 in the nuclear fraction [42] and antibodies against EhPAP that has been previously located in both CE and NE [36]. In some experiments, proteins were analyzed in the presence of a higher concentration of β-mercaptoethanol (8%) and 8 M urea, in order to improve reducing conditions.…”

“…cDNA were synthesized using 1 μg total RNA, 100 ng oligo (dT18), 100 mM DTT, 10 mM dNTPs, 40 U SUPERase-in (Ambion) and 200 U Superscript II reverse transcriptase (Invitrogen) in first-strand buffer, for 1 h at 42°C. Semi-quantitative PCR assays were performed with 1⁄5 volume of the reverse transcription mixture, 10 mM each dNTPs, 5 mM MgCl 2 and 2.5 units of Taq DNA polymerase (Invitrogen) [42], using 5´-GAACAACAGAACCAATGCAAG-3´ (sense) and 5'-CAATTTTTAATCTTCCTCCAACA-3' (antisense) as internal primers for EhCFIm25 transcripts. Amplification was performed as follows: 94 ºC for 5 min; 94 ºC for 1 min, 53 ºC for 1 min and 72 ºC for 1 min, for 25 cycles, and a final extension step at 72 ºC for 7 min.…”

The Role of Lipopeptidophosphoglycan in the Immune Response toEntamoeba histolytica

“…Several genes from E. histolytica were cloned, sequenced and expressed in an effort to identify new drug targets for this parasite, including the alcohol dehydrogenase gene (Ehadh3) [18], the ferredoxin oxidoreductase gene [19], the EhDEAD1 RNA helicase gene [20], and the Ehvma2 gene (which encodes the B subunit of the vacuolar ATPase) [21]. Mechanisms of drug resistance in E. histolytica were also studied, and it was determined that the multidrug-resistant phenotype is regulated at the transcriptional level by the P-glycoprotein-like genes (EhPgp) 1 and 5 [22].…”