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• W2008131010 abstract "Introduction In invasive amebiasis, attempts have been made to correlatethe biochemical and functional properties observed in vitro with the virulence of trophozoites, using as a measure of vir-ulence their ability to produce amebic hepatic abscess in ani-mal models (1). Invasion of host tissues by the trophozoitesis accompanied by contact-dependent cell lysis and phagocy-tosis, as well as by the secretion of lytic components (2). Re-cently, we reported that Entamoeba histolytica contains andsecretes an acid phosphatase (AP), in contrast with Enta-moeba dispar , which possesses the activity but is unable to se-crete it (3). Additionally, we have described the purificationand properties of the E. histolytica acid phosphatase (4). Inthis work, we present data concerning the subcellular local-ization by immunofluorescence of the AP in fixed trophozo-ites, as well as its in situ localization in infected tissue by usingpolyclonal antibodies prepared against purified AP. Materials and Methods Cell culture. E. histolytica HM-1:IMSS strain was grownas described (4). Purification of AP. The enzyme was purified as reportedpreviously (4). Polyclonal anti-AP antibody production. Balb/c mice wereimmunized subcutaneously at least four times with DEAE-enriched fractions. Samples were emulsified with Titer Maxthe first and second times, and with Melox at subsequenttimes. Immunofluorescence analysis. Trophozoites were fixedwith 4% paraformaldehyde for 1 h at 37 8 C and permeabi-lized with PBS-T (0.2% Triton X-100) for 20 min at roomtemperature. Cells were incubated with anti-AP antibodies(1:200), then incubated with a FITC-conjugated secondarygoat-antimouse antibody (1:50), and visualized by confocalmicroscopy. Immunohistochemistry for light and electron microscopydetection of AP. For abscess development, the reportedtechnique was followed (5). After different times postinocu-lation, animals were anesthetized with sodium pentobarbital(94.5 mg/kg of body weight) and the portal vein uncoveredto perfuse the liver with 4% paraformaldehyde, 1% glutaral-dehyde, and 15% picric acid in 0.1 M sodium cacodylatebuffer for 30 min. The liver was dissected and small frag-ments 0.1–0.2 cm thick were transferred to the same fixa-tive and incubated for at least 2 h. For light microscopy,paraffin sections were treated as reported previously (5), us-ing the anti-AP antibody at a 1:100 dilution. For electronmicroscopy, liver samples were included in LR-White, theanti-AP antibody used at a 1:50 dilution, and the secondaryantibody (a 20-nm coloidal gold-labeled goat-antimouseIgG) at a 1:20 dilution (5). Results and Discussion Subcellular localization of E. histolytica AP . To learn moreabout the subcellular localization of AP in trophozoites, weprepared polyclonal monospecific antibodies against AP.DEAE-eluted material was used directly to immunize mice.We found that the antibodies recognized a high-molecular-weight component of approximately 120 kDa present in sol-ubilized fraction (data not shown). Immunofluorescence localization of E. histolytica AP. Thestaining pattern of the anti-AP antibody showed a peripheralstaining in nonpermeabilized cells, whereas in permeabi-" @default.
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• W2008131010 date "2000-07-01" @default.
• W2008131010 modified "2023-10-18" @default.
• W2008131010 title "Subcellular Distribution and In Situ Localization of the Acid Phosphatase of Entamoeba histolytica" @default.
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• W2008131010 doi "https://doi.org/10.1016/s0188-4409(00)00208-3" @default.
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