Biomedical subjects

Stéphane Swillens

Publications and source records attributed to Stéphane Swillens.

5 recordsLinked to original sources

PubMed2006

Limitations and practical procedure in BclII-Ig heavy chain gene rearrangement real-time quantitative polymerase chain reaction.

Follicular lymphoma is characterized by the t(14;18)(q32;q21) translocation, which juxtaposes Ig heavy chain gene (IgH) sequences with the BclII gene. Several publications have highlighted the importance of molecular follow-up in follicular lymphoma, demonstrating that the detection of cells bearing the BclII-IgH rearrangement by real-time quantitative polymerase chain reaction (RQ-PCR) can anticipate a clinical relapse. In this context, we developed a BclII-IgH RQ-PCR. We began with SYBR Green I detection technology but observed that this system does not allow an accurate measurement of the tumor load when working with genomic DNA. While we were designing the assay using Taqman technology, Moppett et al (Moppett J, van der Velden VHJ, Wijkhuijs AJM, Hancock J, van Dongen JJM, Goulden N: Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 2003, 17:268-270) reported PCR inhibition problems in around 15% of blood and bone marrow samples, affecting the DNA quantification and thus the assessment of minimal residual disease. They demonstrated that this PCR inhibition could be partially resolved by adding nonacetylated bovine serum albumin. In our studies, we observed the same phenomenon in a single follicular lymphoma case and extended our study to other available cases. As a result, we suggest a new RQ-PCR procedure that is based on Taqman probe technology and that takes into account the PCR inhibition problems, making this assay more reliable in a routine molecular laboratory.

Aged↗

PubMed2005

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.

Allosteric Regulation↗

PubMed2004

Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve.

Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.

Animals↗

PubMed2003

Bidirectional synaptic plasticity as a consequence of interdependent Ca2+-controlled phosphorylation and dephosphorylation pathways.

Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either long-lasting potentiation (LPT) or depression (LTD). The bidirectional character of synaptic plasticity may result at least in part from an increased or decreased responsiveness of the glutamatergic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) due to the modification of conductance and/or channel number, and controlled by the balance between the activities of phosphorylation and dephosphorylation pathways. AMPA-R depression can be induced by a long-lived Ca2+ signal of moderate amplitude favouring the activation of the dephosphorylation pathway, whereas a shorter but higher Ca2+ signal would induce AMPA-R potentiation resulting from the preferential activation of the phosphorylation pathway. Within the framework of a model involving calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin (PP2B) and type 1 protein phosphatase (PP1), we aimed at delineating the conditions allowing a biphasic U-shaped relationship between AMPA-R and Ca2+ signal amplitude, and thus bidirectional plasticity. Our theoretical analysis shows that such a property may be observed if the phosphorylation pathway: (i) displays higher cooperativity in its Ca2+-dependence than the dephosphorylation pathway; (ii) displays a basal Ca2+-independent activity; or (iii) is directly inhibited by the dephosphorylation pathway. Because the experimentally observed inactivation of CaMKII by PP1 accounts for this latter characteristic, we aimed at verifying whether a realistic model using reported parameters values can simulate the induction of either LTP or LTD, depending on the time and amplitude characteristics of the Ca2+ signal. Our simulations demonstrate that the experimentally observed bidirectional nature of Ca2+-dependent synaptic plasticity could be the consequence of the PP1-mediated inactivation of CaMKII.

Animals↗

PubMed2002

Reduced transition between open and inactivated channel states underlies 5HT increased I(Na+) in rat nociceptors.

We previously demonstrated that activation of a 5HT(4) receptor coupled cAMP-dependent signaling pathway increases tetrodotoxin-resistant Na(+) current (I(Na)) in a nociceptor-like subpopulation of rat dorsal root ganglion cells (type 2). In the present study we used electrophysiology experiments and computer modeling studies to explore the mechanism(s) underlying the increase of I(Na) by 5HT. In electrophysiological experiments with type 2 dorsal root ganglion cells, 5HT increased peak I(Na) and the activation and inactivation rate, without significantly affecting the voltage dependency of activation or availability. Studies on the voltage dependency of channel availability, time course of removal of inactivation, and inactivation of evoked Na(+) currents suggested that there are at least two inactivation states of the Na(+) channel, one (I(fast)) that is induced and retrieved faster than the other (I(slow)). Long (1 s), but not short (60 or 100 ms), inactivating conditioning pulses (CPs) suppressed the 5HT-induced increase in I(Na). Computer modeling studies suggest that 5HT increased I(Na) mainly by decreasing the transition rate (k(OI1)) from an open state to I(fast). Furthermore, 5HT increased I(Na) activation and inactivation rates mainly by increasing the transition rate from closed to open (k(C3O)) and from I(fast) to I(slow) (k(I1I2)), respectively. The antagonism of the 5HT-induced increase in I(Na) by 1-s inactivation CPs may be due an enhancement of transitions from I(fast) to I(slow), via the increase in k(I1I2). This may deplete the pool of channels residing in I(fast), reducing the frequency of reopenings from I(fast), which offsets the increase in I(Na) produced by the reduction in k(OI1). The above findings fit well with previous studies showing that activation of the cAMP/PKA cascade simultaneously increases voltage sensitive tetrodotoxin-resistant Na(+) conductance and inactivation rate in nociceptors. The antagonism of the effects of 5HT by long inactivation CPs suggests that drugs designed to induce and/or stabilize the I(slow) state might be useful for reducing hyperalgesia produced by inflammatory mediators.

Animals↗