Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -0

Non-irradiated (No UV-C) and irradiated (UV-C) trophozoites harvested at different times (30 min, 3, 6 and 12 h). Upper panels, histograms show the DNA fragmentation percentage in fluorescence positive cells. The abscissa indicates fluorescence of propidium iodide (PI), and the ordinate indicates fluorescence of Alexa 488-labeled 3' ends of DNA. The number inside each histogram denotes the percentage of fluorescence positive cells above the cut-off line. Lower panels, PI-staining cells were checked in the epifluorescence microscope to confirming the absence of cytoplasmic stain. PI, propidum iodide, N, Nomanski optics. . Neutral comet assays of non-irradiated (No UV-C) and irradiated (UV-C) trophozoites harvested at different times (30 min, 3, 6 and 12 h). Electrophoretic migration of DNA was from left (anode) to right (cathode).<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -6

Ee probe. Lanes 2 to 4, ssDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, ssDNA incubated with increasing concentrations of mock purified fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (Cto C) are shown by arrowheads. . Partially purified rEhRAD51 was incubated with [α-P]dATP labeled dsDNA and interactions were resolved through PAGE. Lane 1, free probe. Lanes 2 to 4, dsDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, dsDNA incubated with increasing concentrations of mock purified fraction elution fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (Cto C) are shown by arrowheads. . D-loop reactions containing 10,000 cpm of [γ-P]dATP-labeled oligonucleotide, circular dsDNA and 0, 2.5, 5 and 7.5 μg of partially-purified rEhRAD51 (lanes 1 to 4) were incubated at 37°C for 30 min with 2 mM of ATP. Negative controls were performed without homologous dsDNA (lane 5) and with heterologous dsDNA oligonucleotide instead of homologous dsDNA (lane 6), both of them using 7.5 μg of EhRAD51 elution fraction. Reaction products were analyzed by agarose gel electrophoresis, transferred to nylon membranes and visualized through a Phosphor Imager. . Densitometric analysis of D-loop products obtained in C. Results are representative of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -2

D trophozoites harvested at different times (UV-C; lane 2, 0.5 h; lane 3, 3 h and lane 4,12 h). Arrowheads denote the length (bp) of each expected amplified internal fragment, as described in Table 2. . Densitometric analyses of RT-PCR products in A. Pixels corresponding to the rRNA product were taken as 100% in each lane. Data are the mean of three independent assays.<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

TUNEL assays in resveratrol-treated trophozoites.

<p><b>A)</b> TUNEL assays of untreated or IC₅₀ resveratrol-treated trophozoites using dUTP-FITC and visualized under the laser confocal microscope. Nuclei were stained with DAPI. Squares in merge images were magnified in the zoom panels. <b>B)</b> Flow cytometry analysis of dUTP-FITC-treated trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls.</p

Heterodimerization of the <i>Entamoeba histolytica</i> EhCPADH virulence complex through molecular dynamics and protein–protein docking

<p>EhCPADH is a protein complex involved in the virulence of <i>Entamoeba histolytica</i>, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with <i>in silico</i> results.</p

Immunochemistry of livers from hamsters inoculated with virulent trophozoites.

<p>Paraffin sections of livers from hamsters treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146287#pone.0146287.g010" target="_blank">Fig 10</a>. <b>A)</b> Non-challenged hamsters (healthy liver). <b>B)</b> Challenged hamsters treated with ethanol. <b>C)</b> Challenged hamsters treated with resveratrol that did no develop hepatic abscesses. <b>D)</b> Challenged hamsters treated with resveratrol that develop small abscesses. <b>E)</b> Challenged hamsters treated with metronidazole. <b>F)</b> Challenged hamsters treated with ethanol and developed only with the pre-immune serum. <b>G)</b> Parasitic burden quantified in 15 sections of livers from hamsters treated as above.</p

PS externalization produced by resveratrol in trophozoites.

<p><b>A)</b> Confocal microscopy of untreated or IC₅₀ resveratrol-treated trophozoites incubated with Annexin V-FITC and PI. Merge: fluorescence channels and phase contrast. <b>B)</b> Flow cytometry analysis of trophozoites incubated with Annexin V-FITC and PI. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Q1: Trophozoite scheme with nucleus stained (red) representing entrance of PI. Q2: Trophozoite scheme representing PI stained (nucleus) and Annexin V plasma membrane stained. Q3: Trophozoite scheme representing plasma membrane integrity. Q4: Trophozoite scheme representing Annexin V (loss of plasma membrane asymmetry) stained without nucleus stained.</p

Ultrastructure of resveratrol-treated trophozoites.

<p>TEM of untreated, 0.4% ethanol-, IC₅₀ resveratrol- and 0.5 mM H₂O₂-treated trophozoites.</p

Effect of resveratrol in trophozoites morphology.

<p><b>A)</b> Light microscopy images of untreated and IC₅₀ resveratrol-treated trophozoites. <b>B)</b> Size of trophozoites during different incubation times measured in images obtained by light microscopy. Boxes represent 50% of population containing the median of three independent experiments. Bars indicate the maximum and minimum sizes of the other 50%. □ Untreated trophozoites. ■ 0.4% ethanol-treated trophozoites. ■ IC₅₀ resveratrol-treated trophozoites. **p<0.01, ***p<0.001. <b>C)</b> Flow cytometry of size (forward scatter) and granularity (side scatter) of trophozoites incubated 48 h with IC₅₀ resveratrol. R1 represents the gate of trophozoites population selected for these experiments. Untreated and 0.4% ethanol-treated trophozoites were used as controls.</p

Effect of resveratrol administration in hamsters intraportally inoculated with virulent trophozoites.

<p><b>A)</b> Healthy livers: Liver of animals not inoculated with trophozoites B) Ethanol: Livers of intraportally inoculated hamsters (3 x 10⁶ virulent trophozoites), treated with 50 μl of ethanol. Damage after 4 days: Livers of animals inoculated with virulent trophozoites and examined four days after challenge. Resveratrol 2d before and 10 d after challenge: Livers of animals treated with resveratrol (100 mg/Kg diluted in 50 μl of ethanol) each 8 h (2 days before and 10 days after inoculation) and examined ten days after challenge. Resveratrol after 4 d challenge: Liver of animals treated with resveratrol, as above, for ten days, starting four days after challenge when abscesses were already formed <b>B)</b> Damage was evaluated as the weight of the abscesses formed divided by the weight of the whole liver, before the injured areas were removed. As a negative control, animals were not inoculated with trophozoites (healthy liver). As a pharmacological control hamsters were treated with 20 mg/Kg of metronidazole. Values represent the mean ± standard error of liver damage in inoculated animals. n = 7. ***p<0.001.</p