Biomedical subjects

Gilles Uze

Publications and source records attributed to Gilles Uze.

3 recordsLinked to original sources

PubMed2006

Performing quantitative reverse-transcribed polymerase chain reaction experiments.

Quantitative polymerase chain reaction (PCR) is as old as PCR, but it has had to wait for the introduction of real-time PCR instruments to become widely used. These instruments allow monitoring of the PCR reaction on line; they involve the use of a fluorescent probe that allows quantification of the amplified DNA. Different fluorescent formats and different applications have been developed for quantitative PCR, but this chapter focuses on the use of the SYBR Green label for the quantification of specific cDNAs in reverse transcription mixes: RT-PCR. We propose optimal reaction conditions for the reactions to be performed on the different available instruments and discuss the important parameters for setting up experiments: specificity, efficiency, and reproducibility. We also introduce the reader to the problems of relative quantification.

Animals↗

PubMed2006

Inquiring into the differential action of interferons (IFNs): an IFN-alpha2 mutant with enhanced affinity to IFNAR1 is functionally similar to IFN-beta.

Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-alpha2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.

Antiviral Agents↗

PubMed2003

The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway.

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.

Cell Membrane↗