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1.
FIG. 3.

FIG. 3. From: Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection .

EhHMGB1 localization to the nucleus. E. histolytica strain HM1:IMSS was transfected with full-length recombinant EhHMGB1 as described in Materials and Methods. The parasites were fixed to glass coverslips, and EhHMGB1 was detected using anti-Myc antibody. Nuclei were stained with DAPI. Images were captured using a confocal microscope (Zeiss). (A) DAPI-stained nuclei (blue); (B) EhHMGB1 (red); (C) bright-field image; (D) colocalization. Results were similar with anti-Strep II (IBA) antibody (data not shown).

Mayuresh M. Abhyankar, et al. Eukaryot Cell. 2008 September;7(9):1565-1572.
2.
FIG. 2.

FIG. 2. From: Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection .

DNA bending and ligase-mediated circularization assay with wild-type (A) or mutant (B) recombinant EhHMGB1. End-labeled 123-bp DNA fragments were preincubated with increasing amounts of wild-type or mutant EhHMGB1 as indicated, followed by ligation with T4 ligase as described in Materials and Methods. Exonuclease III digestion was used to verify the production of exonuclease III-resistant DNA circles. The positions of linear and circular DNAs are shown. The bands above the position of circular DNA were exonuclease III-sensitive linear multimers. Abbreviations: WT, wild type; DM, double mutant; Δacid, C-terminal tail deletion mutant.

Mayuresh M. Abhyankar, et al. Eukaryot Cell. 2008 September;7(9):1565-1572.
3.
FIG. 4.

FIG. 4. From: Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection .

Recombinant EhHMGB1 enhanced DNA binding by p53. Autoradiogram of a representative EMSA showing the effects of increasing concentrations of EhHMGB1 or its mutants on the binding of p53 to its target. Human recombinant p53 (150 ng) and increasing amounts of wild-type or mutant EhHMGB1 recombinant proteins (0.8 to 2.4 μM) were incubated with 32P-end-labeled oligonucleotide containing the GADD45 p53 binding site. Lane 1 contained probe alone. The arrow indicates the location of the DNA-p53 complex. The smear, denoted by a brace, indicates the location of the DNA-protein complex due to non-sequence-specific binding of wild-type or mutant EhHMGB1 to the probe. Note that the double mutant (DM) failed to bind DNA whereas the Δ-Acid mutant apparently shows increased nonspecific binding to the probe compared to that for wild-type protein.

Mayuresh M. Abhyankar, et al. Eukaryot Cell. 2008 September;7(9):1565-1572.
4.
FIG. 1.

FIG. 1. From: Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection .

Multiple sequence alignment of HMGB domain-containing proteins. The boxed residues represent conserved amino acids important for HMGB1 function. A polar amino acid, such as serine or threonine, and a hydrophobic residue at these two positions, respectively, typically signify a non-sequence-specific HMG protein in eukaryotes. The bold residues represent the HMGB domain of EhHMGB1, whereas the underlined residues represent the C-terminal acidic tail. Abbreviations are as follows: SJ, Schistosoma japonicum (accession number AY223388); SM, Schistosoma mansoni (accession number AY485339); DM, Drosophila melanogaster (accession number NM_166480); RN, Rattus norvegicus (accession number XM_001058770); PF1, Plasmodium falciparum HMGB1 (accession number XM_001350402); PF2, Plasmodium falciparum HMGB2 (accession number XM_001349310); EH, Entamoeba histolytica HMGB1. The CLUSTAL program was used to align either full-length proteins (S. japonicum, S. mansoni, D. melanogaster, P. falciparum HMGB1 and HMGB2, and E. histolytica) or HMGB B domain alone (R. norvegicus) from various species. The amino acid numbers are shown to the right. *, identical amino acids; “:,” strongly similar amino acids; “.,” weakly similar amino acids; “-,” gap in the sequence.

Mayuresh M. Abhyankar, et al. Eukaryot Cell. 2008 September;7(9):1565-1572.
5.
FIG. 5.

FIG. 5. From: Characterization of an Entamoeba histolytica High-Mobility-Group Box Protein Induced during Intestinal Infection .

Microarray analysis of strain overexpressing EhHMGB1. (A) Venn diagram showing overlap of genes differentially modulated upon EhHMGB1 overexpression in vitro and in the mouse model of amebiasis on days 1 and 29 (in vivo) as described by Gilchrist et al. (27). Thirty-three transcripts were significantly modulated upon EhHMGB1 overexpression, of which 60% were also modulated at either day 1 or day 29 in the mouse model of amebiasis. EhHMGB1 was upregulated at both time points (two- and sixfold, respectively), although a statistically significant change occurred only at day 1. The observed overlap between the modulated mRNA in vivo and the transcripts changed in vitro upon EhHMGB1 overexpression is much greater than would be expected by chance (P < 0.0001 by chi-square test). (B) Effect of EhHMGB1 overexpression in vitro. Gene annotation is shown on the x axis (including the important virulence factors EhCP-A7 and Lgl3); the change in either a positive or a negative direction is indicated on the y axis. Black bars and gray bars indicate the changes that occurred in trophozoites isolated from the mouse model of amebiasis (in vivo) at day 1 and day 29, respectively (27). White bars indicate the modulation that occurred in strains overexpressing EhHMGB1. Abbreviations: AMA, alpha-amylase; MGL, methionine gamma-lyase; DLP, dynamin-like protein; ZFP, zinc finger protein; SULT, sulfotransferases; GLY, glycosyltransferase; MP, membrane protein.

Mayuresh M. Abhyankar, et al. Eukaryot Cell. 2008 September;7(9):1565-1572.

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