Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients

Intracellular transport of GPI-anchored proteins by yeast

We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas 1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas 1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas 1p. The need for ceramide can be overcome in the mutant strain