Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia.

Myb family transcription factors are important in regulating cell proliferation, differentiation, and cell cycle progression. Giardia lamblia differentiates into infectious cysts to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. We have identified an encystation-induced Myb2 protein, which binds to the promoter regions of the cwp genes and myb2 itself in vitro. To elucidate the role of Myb2 in G. lamblia, we tested the hypothesis that Myb2 can activate encystation-induced genes. We found that overexpression of Myb2 resulted in an increase of expression of CWP1 at both protein and mRNA levels. Interestingly, the Myb2-overexpressing trophozoites had increased capability to differentiate into cysts. In cotransfection assays, Myb2 was able to transactivate the cwp promoters and its own promoter in vivo, suggesting that its gene can be positively autoregulated. Moreover, deletion of the N- or C-terminal domain resulted in a decrease of transactivation and autoregulation function of Myb2. We also found that the promoter of a newly identified encystation-induced gene, the giardial myeloid leukemia factor-like gene, has the Myb2 binding sites and that its mRNA levels were increased by Myb2 overexpression. Chromatin immunoprecipitation assays confirmed that Myb2 was bound to the promoters with its binding sites. Transfection of the myb2 antisense construct reduced the levels of the cwp1 transcripts and cyst formation. Our results suggest that Myb2 is a potent transactivator of the cwp genes and other endogenous genes and plays an important role in G. lamblia differentiation into cysts.

Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection.

Two systems for stable transfection of Giardia have been established using selection either by neomycin or by puromycin. We asked if these selection systems themselves influenced expression of endogenous giardial genes. Northern blot analysis showed a approximately 1.4 to approximately 7-fold increase in the encystation-induced cyst wall protein 1 (cwp1), cwp2, and gmyb2 gene transcripts in the drug selected cell lines during vegetative growth, compared with untransfected cells. However, the levels of the constitutive ran, lrp3, or alpha2-tubulin gene transcripts decreased slightly or did not change in these stably transfected cell lines. Part of the effect could be due to drug selection, since treatment of untransfected cells with G418 or puromycin also had similar effects. Nuclear run-on assays showed that part of the effect comes from an increase in transcription initiation rate. The levels of CWP and cyst formation during vegetative growth also increased in the transfected cell lines. Using proteomic technologies, we identified eight genes whose expression is upregulated in neomycin selected cell lines, including phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, ornithine carbamoyltransferase, carbamate kinase, orf 16424, cyclophilin, co-chaperone-like p21, and bip. Six of these are also upregulated in puromycin selected cell lines. Our results indicate that transfection and drug selection, per se, can alter expression of genes involved in metabolism, protein folding, and differentiation status in Giardia.

Microglia-derived glial cell line-derived neurotrophic factor could protect Sprague-Dawley rat astrocyte from in vitro ischemia-induced damage.

The interrelationship between microglia and astrocytes in cerebral ischemia was determined in vitro by adding in vitro ischemia-induced supernatant from microglia into astrocytes under the same conditions (glucose-, oxygen- and serum-free). The involvement of glial cell line-derived neurotrophic factor (GDNF) was further investigated by immunoblocking assay and Western blot analysis. Results showed that microglia-derived supernatant protected against in vitro ischemia-induced damage of astrocytes and this protection was pre-blocked by anti-GDNF but not normal rabbit serum. In addition, in vitro ischemia appeared to induce the expression of GDNF in microglia. These results indicate that microglia-derived protection on astrocytes during in vitro ischemia is GDNF-dependent.