We were not able to corroborate this previous result, and in fact, we demonstrated that gene expression remains consistently elevated during ring differentiation, becoming drastically reduced in every isolate when parasites reached the trophozoite stage. by VX-702 visual scoring of VX-702 at least 100 infected erythrocytes stained with the immune serum (B). Shown is the percentage of protein associated with the erythrocyte membrane (EM, blue), Maurers clefts (MC, violet), parasite membrane and parasitophorous vacuole membrane complex (PM/PVM, red), and inside the parasitic boundary (parasite, grey); cells that lacked specific fluorescence signals are also shown (negative, white). The summary percentage of all location sites is greater than 100 because some proteins localized to multiple sites within one cell. n.d.: not determined.(TIF) pone.0049540.s003.tif (2.3M) GUID:?F3CE4722-CBFB-4296-AA09-03AC28F75350 Figure S4: IFA and quantification of VSA localization in Mouse monoclonal to HRP isolates #1C4 and 3D7 using the -ATS antiserum. IFA of different parasitic stages, as determined by time of cultivation (h) using an antiserum directed against the C-terminal domain of cultivation (h) using an antiserum directed against A-type RIFIN proteins (-RIF29n) (A). Fluorescence signals and localization was quantified by visual scoring of at least 100 infected erythrocytes stained with the -RIF29n serum (B). Shown is the percentage of protein associated with the erythrocyte membrane (EM, blue), Maurers clefts (MC, violet), parasite membrane and parasitophorous vacuole membrane complex (PM/PVM, red), and inside the parasitic boundary (parasite, grey); cells that lacked specific fluorescence signals are also shown (negative, white). The summary percentage of all location sites is greater than 100 because some proteins localized to multiple sites within one cell. n.d.: not determined.(TIF) pone.0049540.s005.tif (2.1M) GUID:?47A1749E-F73E-40FA-A40F-75A1D501ED79 Figure S6: IFA and quantification of VSA localization in isolates #1C4 and 3D7 using the -RIF40 antiserum. IFA of different parasitic stages, as determined by time of cultivation (h) using an antiserum directed against A-type RIFIN proteins (-RIF40) (A). Fluorescence signals and localization was quantified by visual scoring of at least 100 infected erythrocytes stained with the -RIF40 serum (B). Shown is the percentage of protein associated with the erythrocyte membrane (EM, blue), Maurers clefts (MC, violet), parasite membrane and parasitophorous vacuole membrane complex (PM/PVM, red), and inside the parasitic boundary (parasite, grey); cells that lacked specific fluorescence signals are also shown (negative, white). The summary percentage of all location sites is greater than 100 because some proteins localized to multiple sites within one cell. n.d.: not determined.(TIF) pone.0049540.s006.tif (2.3M) GUID:?48E75BD8-0A5D-4D60-BC14-937E560BD419 Figure S7: IFA and quantification of VSA localization in isolates #1C4 and 3D7 using a mixture of -STEVOR antisera. IFA of different parasitic stages, as determined by time of cultivation (h) using an antisera mixture directed against different STEVOR variants (-STEVOR-mix) (A). Fluorescence signals and localization was quantified by visual scoring of at least 100 infected erythrocytes stained with the -STEVOR-mix (B). Shown is the percentage of protein associated with the erythrocyte membrane (EM, blue), Maurers clefts (MC, violet), parasite membrane and parasitophorous vacuole membrane complex (PM/PVM, red), and inside the parasitic boundary (parasite, grey); cells that lacked specific fluorescence signals are also shown (negative, white). The summary percentage of all location sites is greater than 100 because some proteins localized to multiple sites within one cell. n.d.: not determined.(TIF) pone.0049540.s007.tif (2.2M) GUID:?FA0F02F4-7B54-4E40-B95A-7F1EA53BD212 Figure S8: IFA and quantification of VSA localization in isolates #1C4 and 3D7 using the – cultivation (h) using an antiserum directed against the semi-conserved domain of cultivation (h) using an antiserum directed against the C-terminal domain of possesses a number of multi-copy gene families, including and which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by in the human host. To investigate the expression of the and family genes under conditions that mimic more closely the natural course of infection, clinical isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns VX-702 in the clinical isolates at various time points during the first intraerythrocytic developmental cycle were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families and at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen VX-702 families were overexpressed in the clinical isolates relative to 3D7, with the exception of the family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins. Introduction During erythrocyte schizogony, the.
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