Anne-Cécile Ribou

An example of molecular co-evolution: Reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.

Native Fluorescence and Mag-Indo-1-Protein Interaction as Tools for Probing Unfolding and Refolding Sequences of the Bovine Serum Albumin Subdomain in the Presence of Guanidine Hydrochloride

Changes in the fluorescence spectrum of tryptophans Trp 134 and Trp 212 in bovine serum albumin (BSA) and of Trp 214 of human serum albumin in the presence of the chaotropic agent guanidine hydrochloride (Gnd) were studied. A detailed analysis of the fluorescence spectrum of native BSA yielded the fluorescence spectrum for each tryptophan of BSA. Modifications in the binding of Mag-indo-1 to BSA, which results in a specific quenching of the fluorescence spectrum of Trp 134 associated with an energy transfer from Trp 134 to the protein-bound Mag-indo-1, were also investigated. Changes occurring when the Gnd concentration is decreased stepwise cover a larger concentration scale of Gnd than the reverse protocol, allowing one to suggest that the resulting conformational changes in the subdomain IA of BSA involve at least three different steps.

Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.

Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.

Lifetime of Fluorescent Pyrene Butyric Acid Probe in Single Living Cells for Measurement of Oxygen Fluctuation

We study the fluorescence lifetime of the well‐known 1‐pyrene butyric acid (PBA) to assess oxygen concentrations in living cells. The behavior of the probe is first studied in water, ethanol, protein solution and liposome suspension. The Stern‐Volmer plot of these solutions is linear, and the bimolecular reaction rate constant agrees with previous observations. In single living cells, the PBA lifetime decreases with oxygen concentration (185 to 55 ns). The probe lifetime differences between living cells and liposome suspension, especially under nitrogen atmosphere, suggest a supplemental pathway for the deactivation of the probe. We simplify further the complex living cells system by stopping the cell functions and studying freshly fixed cells. In this case, we obtained an increase of PBA lifetime under nitrogen atmosphere (215 ns).

Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells.

We investigate the relative importance of the different mechanisms of Adriamycin, an anthracycline, and their interrelations, in particular the link between cell cycle arrest, cell death, and generation of reactive oxygen species (ROS) that is suspected to be the origin of cardiotoxic side-effects. We introduced a lifetime fluorescence based technology and used videomicrofluorometry, two efficient analytical methods. We show that depending on the doses and time after incubation, ADR will not reach the same compartments (nucleus, mitochondria, cytosol) in the cells, having consequences on the production of ROS, growth arrest pathways and cell death pathways.

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: A comparison between hypoxic and normoxic cells

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O2) and normoxic (21% O2) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.

Measurements of calcium with a fluorescent probe Rhod-5N: Influence of high ionic strength and pH

We describe a new method for the spectroscopic determination of high calcium concentration using a fluorescent probe Rhod-5N. This method was investigated in order to be utilized in high ionic strength solution, such as seawater. The probe is fluorescent when bound to calcium, LM, but not as the free form L. The dissociation constant of the equilibrium (0.14mM) was determined at several ionic strengths, i.e. in the absence and in the presence of additional ions (0.7M NaCl). The influence of pH was studied. In order to correctly model the experimental data, we included a new fluorescent compound: LHM (calcium bound protonated probe). The first acidity constant (0.02muM) and the second dissociation constant (4.5mM) were calculated. A useful range for the determination of calcium concentration is provided. Such a method is fast and easy to carry out.

Effect of Adriamycin treatment on the lifetime of pyrene butyric acid in single living cells

We investigated the fluorescence lifetime of pyrene butyric acid (PBA) using various O2 concentrations in cells. Both in living and freshly fixed cells, PBA lifetime decreased with oxygen concentration. We recorded decay curves in single cells and measured PBA lifetime and NAD(P)H intensity values. Under nitrogen atmosphere, the probe lifetime differences (199 and 209 ns in living and freshly fixed cells, respectively) suggest a supplemental pathway for the deactivation of the probe when the cell functions are not stopped. We propose reactive oxygen species (ROS) to be the additional quenchers that cause this decrease. We further studied the effect of drugs generating ROS the anthracycline doxorubicin (adriamycin). For living cells, PBA lifetime decreased after adriamycin (ADR) treatment (200 and 1000 ng/ml). This supports our hypothesis that under nitrogen atmosphere and for freshly fixed cells, PBA lifetimes increase to an unchanging value due to absence of quenchers.

Time-resolved microfluorimetry: An alternative method for free radical and metabolic rate detection in microalgae

Oxidative stress leads to an increase in the production of reactive oxygen species in cells and can be induced by environmental factors. To study free radical production in living microalgae, we use time-resolved microfluorimetry, a technology adopted from research on mammalian cells. In contrast to fluorescent probe-based measurements that rely on intensity changes, our sensor detects the presence of free radicals through collisional quenching, and is insensitive to most artifacts commonly observed with intensity-based methods. A new probe, 1-pyrenebutanol allows estimation of free radicals production in the green microalga Tetraselmis ssp., for the first time. In addition, our method monitors simultaneously metabolic rate (through bound-free NAD(P)H ratio). Our results show that free radical production in algal cells is correlated to algal aging, and that during cell growth phases both intracellular free radicals and metabolic activity increase. Concerning thermal stress, we observe that rapid and low temperature changes (<10°C) induce instantaneously an increase in ROS production. Our findings provide new insights into the production of free radicals in response to environmental stresses in unicellular green algae.

Mag-indo-1 as a potential reporter of the 3D conformation of protein subdomains

Mag-indo-1 is a well known fluorescent probe. Magnesium complexation results in a shift of the emission fluorescence spectrum from 480 nm to 417 nm with an intensity proportional to the magnesium concentration in the range 0.6 to 30 mM. Although designed as a specific magnesium chelator, Mag-indo-1 is also able to bind calcium and zinc. All these cationic interactions induced the same spectral shift but the fluorescence intensity and the dissociation constant are dependent of the nature of the cation. Furthermore Mag-indo-1 can also bind proteins through a specific interaction with some histidin residues. That interaction induces a characteristic spectral shift of the emission fluorescence spectra from 480 to 457 nM. All these properties suggest that Mag-indo-1 could be used to study the protein-cation binding. Emission and synchronous fluorescence techniques have been used to monitor that interaction with proteins such as bovine serum albumin, human serum albumin, turkey white egg lysozyme. Using a method of resolution of complex fluorescence spectra, it has been possible to calculate the number of interaction sites and the correlative dissociation constants. Depending on the nature of the protein a quenching of the natural fluorescence of the protein was observed, associated with an energy transfer from some tryptophan(s) to Mag-indo-1. All these data were tentatively correlated with the available information on the 3D conformation of the proteins. These results suggest that Mag-indo-1 could be used as an intramolecular fluorescent ruler to monitor the changes in 3D conformation of specific sub-domains of proteins.