Toxoplasma gondii: expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages.
In this study, effects of GRA1 organelle-targeted expression on macrophage functions were investigated. The recombinant plasmid pCMV/myc/ER-GRA1 was constructed and then was transfected into murine macrophage RAW264.7 by Lipofectamine, selected by resistance of G418. The selected mono-clone cell line was named ER-GRA1-RAW264.7. The expression of GRA1 was localized in ER of ER-GRA1-RAW264.7 cells by indirect immunofluorescence detection. GRA1 mRNA expression level in ER-GRA1-RAW264.7 cell was significantly enhanced with a concomitant increase in its growth and adherence activity. Fluorescence intensity of intracellular calcium in ER-GRA1-RAW264.7, ER-ctrl-RAW264.7 and RAW264.7 cells in the presence of 1 mmol/l arachidonic acid (AA) were assayed by confocal microscopy using calcium-sensitive dye, Fluo-3 AM. Cytoplasm [Ca2+]i peaked at about 18 s after AA treatment, and cytoplasm [Ca2+]i of RAW264.7 cell almost instantly stepped up after AA was added, and peaked in 3 s, with a minor cytoplasm [Ca2+]i vibration subsequently. These results demonstrated that the expression of GRA1 in ER of macrophages promotes both growth and adherence of macrophages and modulates the intracellular calcium release stimulated by AA.
Lin
Jie
J
Research Center of Molecular Medicine, Fujian medical University, Fuzhou, 350004 Fujian Province, PR China.
Lin
Xu
X
Yang
Guo-hua
GH
Wang
Yong
Y
Peng
Bi-wen
BW
Lin
Jian-yin
JY
D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis / Q000235:genetics*; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D004274:DNA, Recombinant / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D004721:Endoplasmic Reticulum / Q000276:immunology*; D015870:Gene Expression; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000469:parasitology*; D008297:Male; D051379:Mice; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014122:Toxoplasma / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D000953:Antigens, Protozoan; D004274:DNA, Recombinant; C412913:GRA1 antigen; D012333:RNA, Messenger; D016718:Arachidonic Acid; D002118:Calcium