45 research outputs found
Evolution of each transmembrane spanindividually.
<p><b>A:</b> Evolutionary tree using the pool of individual TMS sequences. A maximum likelihood method based on the JTT matrix-based model was used, with 500 bootstrap replicates. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The tree with the highest log likelihood (−3119.4504) is shown. Two major superclusters are separated by a solid line in which numbers indicate the main positions for the grouped TMSs. Colored numbers indicate consecutive TMSs from each supercluster. Scale bar corresponds to distance expressed in amino acid substitutions per site. <b>B:</b> Similarity as a function of position was plotted to compare TMSJ against S-TMSJ, and LJ against S-LJ.</p
Hexose transporters synteny analysis.
<p>The genomic contexts of the <i>Trypanosoma</i> spp and <i>Leishmania</i> spp hexose transporters were analyzed. The figure represent one syntenic orthologous group (highlighted in green) comprising the genes LinJ.36.6550 (and its paralog LinJ.36.6560), LmjF.36.6280 (and its paralogs LmjF.36.6290-LmjF.36.6300, called lmgt1-3), LmxM.36.6280 (and its paralogs LmxM.36.6290-LmxM.36.6300), TcIL3000.10.7320, and Tb427.10.8450 (and its paralog Tb427.10.8530, called THT1). This group of genes has expanded into 10 tandem duplications. Lila shadows indicates the syntenic genes and all the sequences are represented by their systematic ID. The synteny analyses and scheme was made at the TriTrypDB (<a href="http://tritrypdb.org/tritrypdb" target="_blank">http://tritrypdb.org/tritrypdb</a>).</p
Phylogeny of the hexosetransporters.
<p><b>A:</b> Evolutionary tree constructed using the full-length amino acid sequences of the kinetoplastid hexose transporters. Sequences coding for glucose transporters of <i>Homo sapiens</i>, <i>Drosophila melanogaster, Danio rerio</i> and <i>Escherichia coli</i> were used as outgroups to root the tree. A maximum likelihood method based on the JTT matrix-based model was used, with 500 bootstrap replicates. The tree with the highest log likelihood (−7654.7770) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The kinetoplastid hexose transporters segregate into two main groups, which correspond to the genera <i>Trypanosoma</i> and <i>Leishmania</i>. <b>B:</b> Schematic representation of the structure of the sequences used. <b>FL:</b> full-length sequence, <b>TMS:</b> transmembrane span, <b>L:</b> loops connecting the TMSs, <b>TMSJ:</b> sequence containing contiguously ordered TMSs, <b>LJ:</b> sequence containing contiguously ordered Ls, <b>S-TMSJ:</b> scrambled TMSJ, <b>S-LJ:</b> scrambled SJ.</p
Evolutionary trees of transmembrane spans and loops.
<p>Evolutionary tree using the TMSJ (<b>A</b>) or LJ (<b>B</b>) sequences. A maximum likelihood method based on the JTT matrix-based model was used, with 500 bootstrap replicates. Trees with the highest log likelihood (−3067.0851 and −4129.5431, respectively) are shown. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site. In both cases, sequences segregate into two main groups corresponding to the genera <i>Trypanosoma</i> and <i>Leishmania</i>. <b>C:</b> The mean distances for each group of sequences analyzed (FL, TMS, TMSJ, S-TMSJ, NT, LJ and S-LJ) were calculated and are presented as similarity values.</p
Description of the main characteristics of the used sequences.
<p><b>AN:</b> Accession Numbers; <b>TMS:</b> number of transmembrane spans in each sequence; <b>NTP:</b> N-terminal position; <b>CTP:</b> C- terminal position; <b>NTL:</b> N- terminal length; <b>I:</b> intracellular; <b>E:</b> extracellular.</p
Chemical structures of Memantine, Amantadine and MK-801.
<p>Chemical structures of Memantine, Amantadine and MK-801.</p
Effect of Memantine on the intracellular cycle of <i>Trypanosoma cruzi</i>.
<p>Panel A: viability of CHO-K<sub>1</sub> cells treated with different concentrations of Memantine (range 50 µM to 1 mM). Viability was assessed by MTT assay. Panel B: effect on the infectivity of trypomastigotes treated only during the period of infection (50–400 µM). Panel C: effect of treatment after invasion of parasites in CHO-K<sub>1</sub> cells (5–300 µM). Panel D: effect of Memantine on intracellular stages. Cells were treated at different stages with 31 µM Memantine (IC<sub>50</sub> value): <b>T</b> (trypomastigote cell invasion), <b>A</b> (amastigote) and <b>IE</b> (intracellular epimastigote-like) stages. In all experiments, we evaluated the burst of trypomastigotes on the fifth day post-infection by counting parasites in a Neubauer chamber. Tukey test: *: p<0.05; **: p<0.01; ***: p<0.001.</p
Forward and side scattering values for epimastigotes treated or not with Memantine.
1<p>Geometrical mean of scatter values of epimastigotes treated or not with 172.6 µM Memantine.</p
Quantification of H<sub>2</sub>O<sub>2</sub>, Ca<sup>2+</sup> and ATP levels in <i>T. cruzi</i>.
<p>Panel A: <b>H<sub>2</sub>O<sub>2</sub> levels</b>, parasites treated with Memantine (172.6 µM) or not treated (control) for 24 hours. After this period, the parasites (1.0×10<sup>7</sup>) were incubated with 25 µM amplex red, and 0.05 U mL<sup>−1</sup> horseradish peroxidase and analyzed on a fluorometer (λ<sub>ex</sub> 563 nm and λ<sub>em</sub> 587 nm). Panel B: <b>Ca<sup>2+</sup> levels</b>, parasites were treated for 4 days and incubated with Fluo-4 AM (5 µM) for 1 hour at 28°C, washed twice in HEPES-glucose and evaluated on a fluorometer (λ<sub>ex</sub> 490 nm and λ<sub>em</sub> 518 nm). Panel C: <b>ATP levels</b>, parasites were treated for 30 hours, and the levels of ATP were assessed using a bioluminescent assay kit (Sigma-Aldrich) and analyzed on a luminometer (λ 570 nm). <i>T</i> test: *: p<0.05; **: p<0.01; ***: p<0.001.</p
<i>Trypanosoma cruzi</i> epimastigote forms, 4<sup>th</sup> day of growth.
<p>Panel A and B: untreated parasites. Panel C and D, parasites treated with Memantine (172.6 µM).</p