Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers.

Quantitative RT-PCR using LUX primers was performed to determine the expression patterns of various transcripts in samples of pluripotent, mouse P-19 stem cells. The P-19 cells were used because they transform into neuron-like cells upon retinoic acid treatment. The expression of neural and stem cell genes, including GLUR1, GABA-B1a, NMDA1, GAP-43, ChAT, BDNF, nestin, BMP-2, BMP-4, and EGR1, was increased, approximately 10- to 1000-fold, during the course of differentiation from 0 to 11 days after induction with retinoic acid. A 3-fold serial dilution of in vitro-transcribed ChAT mRNA from 66 to 10(7) copies was discriminated by qRT-PCR using fluorogenic LUX primers. Results of quantitation using PCR utilizing dual LUX primer pairs were similar to quantitation using single LUX primers, and to results derived by using an alternate method for qRT-PCR, the 5(')-nuclease probe assay. The efficiencies of PCRs using various primer sets were similar, so that a comparative C(T) method of quantifying relative amounts of transcripts was performed. We conclude that real-time RT-PCR using fluorogenic LUX primers is a reliable, effective alternative to present methods for quantifying several transcripts in neural stem cells.

Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability.

Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide-phosphate repeat unit Galbeta1,4Manalpha1-PO4, shared by other developmentally regulated molecules implicated in parasite virulence. Functional complementation of a Leishmania donovani LPG-defective mutant (OB1) accumulating a truncated LPG containing only the Manalpha1-PO4 residue of the first repeat unit identified LPG3, the Leishmania homolog of the mammalian endoplasmic reticulum (ER) chaperone GRP94. LPG3 resembles GRP94, as it localizes to the parasite ER, and lpg3(-) mutants show defects including down-regulation of surface GPI-anchored proteins and mild effects on other glycoconjugates. LPG3 binds cellular proteins and its Leishmania infantum GRP94 ortholog is highly immunogenic, suggesting a potential role in directing the immune response. However, null lpg3(-) mutants grow normally, are completely defective in the synthesis of phosphoglycans, and the LPG3 mRNA is regulated developmentally but not by stress or heat. Thus the role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes. Like the other glycoconjugate synthetic pathways in this parasite, its activity is focused on molecules implicated in virulence rather than viability.