Françoise Rabaste

Intracellular pH of Candida albicans blastospores as measured by laser microspectrofluorimetry and 31P-NMR

The intracellular pH (pHi) of Candida albicans blastospores harvested from 8 h or 48 h cultures was determined under identical experimental conditions by two different techniques: 31P-NMR and laser microspectrofluorimetry. Time dependence of pHi was monitored by 31P-NMR on the whole cell population. Microspectrofluorimetry, after loading of the cells with SNARF-1, enabled the determination of pHi in isolated cells and its distribution among the cell population. By this method, the vacuolar pH could not be distinguished from the cytoplasmic pH in C. albicans blastospores, but alkalization of pHi was observed at the beginning of germ tubes. The absolute values of pHi determined by 31P-NMR were slightly different from those obtained by laser microspectrofluorimetry. However, the pH distributions in the cell population were converging. For blastospores in exponential phase a gaussian distribution of pHi was observed with both methods, the cells maintained a steady pHi value when the external pH was varied from 5.5 to 8.5. For cells in stationary phase two pools were identified: the combination of the two techniques demonstrated the presence of two different subpopulations. One of these population (with lower pH) was able to commute to the other one with time as shown by 31P-NMR kinetics. This information is reported here for the first time in C. albicans.

Modifications of pH and K + gradients in Candida albicans blastospores induced by Amphotericin B. A 31p NMR and K + atomic absorption study

Candida albicans blastospores harvested from 8 h (exponential) or 48 h (stationary) cultures were incubated with increasing doses of amphotericin B (AmB). The time course of H+ influx and K+ efflux was monitored by in vivo 31P NMR and K+ atomic absorption respectively. AmB was shown to be more active on exponential phase cells than on stationary phase cells. For both growth phases, K+ leakage occurred before pH acidification. In light of these results, together with iodoacetate experiments, it seems difficult to assert that K+ leakage is a secondary effect resulting from an increase in the permeability to protons, as formerly proposed. In addition, no H+ over K+ selectivity of pores formed by AmB could be detected. Finally, some unexpected results were afforded by 31P NMR experiments: a broadening of Pi signals was detected on exponential phase cell spectra when the blastospores were incubated with 10(-3) and 10(-4) M AmB reflecting a transient heterogeneity of the intracellular pH within the cell population. For stationary phase blastospores, two subpopulations (IIa and IIb) were detected; population IIb, with a more acidic pHi, was much more sensitive to AmB action.

X-Ray Diffraction to Study the Oxidation Mechanism of Chromium at Elevated Temperatures

It is demonstrated that the oxidation behaviour of chromium was significantly different according to the temperatures, i.e. 800, 900, and 1000 °C. Under isothermal condition, the formation of a chromia scale on pure PM chromium follows parabolic kinetics, indicative of a diffusion-controlled growth mechanism. At each temperature the mass gain curves showed some discontinuities that can be explained by the formation of cracks, that gives a direct access to nonoxidized metallic surfaces. Nevertheless, under the experimental conditions, chromium was able to form a rather protective oxide scale, preventing the underlying substrate against severe corrosion by a healing process. Cross-section examinations revealed that under the oxide layer a nitrogen-rich sub-surface layer was formed in the substrate. X ray diffraction results prove that due to inward diffusion of nitrogen, a solid solution of nitrogen in chromium was formed, which finally reacts with each other forming chromium nitrides CrN and Cr2N. The presence of this nitrogen rich layer changes the metallic matrix hardness and then a decrease of the oxide scale adherence was observed.

Na+ and K+ transport by 4-chlorophenylurethane-monensin in Enterococcus hirae de-energized and energized cells studied by 23Na-NMR and K+ atomic absorption

Na+ and K+ movements induced by 4-chlorophenylurethane-monensin, which presents an inverted ion selectivity (K+ > Na+) in model systems compared with monensin, were followed on Enterococcus hirae cells by 23Na-NMR and K+ atomic absorption. For de-energized cells, the urethane derivative is much more selective for K+ than monensin, but only at low concentrations (10(-3)-10(-4) mM). For higher concentrations, as previously shown for monensin, the sodium and potassium movements are driven by the ion gradients present. On energized cells, both K+ and Na+ gradients were highly perturbed, and this can be related to the higher toxicity in mice and bacteria for this derivative.

Phosphate-dependent sodium transport in S. faecalis investigated by 23Na and 31P NMR.

Na+ movements in S. faecalis were studied by 23Na NMR. They proved to be dependent on phosphate concentration in the buffer during the de-energization step. K+ and H+ were also studied respectively by potentiometry and 31P NMR and were shown not to be implicated. For de-energized cells the internal phosphate concentration, on the contrary, was directly linked to the external phosphate contained in the buffer. The experiments showed a Na+/Pi dependence in this prokaryote so far known only in eukaryotes.

Effect of Lanthanum Sol-Gel Coating on the Oxidation Behaviour of the AISI 304 Steel at 1000°C

The aim of the present work is to investigate the effect of Lanthanum surface addition on the oxidation behaviour of the AISI 304 stainless steel, in air, at 1000°C. The in situ X-ray diffraction (XRD) analyses on the blank steel reveal that after the first 10h oxidation, a change in the structural composition of the oxide scale occurs. During the first ten hours oxidation an initial growth of chromia and Mn1,5Cr1,5O4 is observed. After 10 h oxidation, chromia is not detected anymore and iron-containing oxides such as hematite (Fe2O3) and iron chromite (FeCr2O4) are observed in the outer part of the scale. With blank AISI 304 specimens, the iron-containing oxides are generally not very protective and show severe spallation during cooling to room temperature due to thermal stresses. They do not allow a good adherence of the corrosion layer under thermal cycling. On the Lanthanum coated AISI 304 Stainless Steel the oxidation rate is 10 times lower. In situ XRD analyses show the absence of iron containing oxides. It reveals the formation of a fine convoluted Cr2O3 layer associated with the formation of the mixed oxides Mn1,5Cr1,5O4 and LaCrO3. LaCrO3 is found to be located at the oxide/steel interface. Our results show that, even though the scale formed under isothermal conditions is not composed of iron containing oxides, Lanthanum sol-gel coating does not prevent spallation during thermal cycling at 1000°C.

Manganese effect on isothermal high temperature oxidation behaviour of AISI 304 stainless steel

Chromia-forming steels are excellent candidates to resist to high temperature oxidizing atmospheres because they form protective oxide scales. The oxide scale growth mechanisms are studied by exposing AISI 304 stainless steel to high temperature conditions in air, and the analyses were carried out by means of thermogravimetry and in situ X-rays diffraction. The in situ XRD analyses carried out during high temperature AISI 304 steel oxidation in air reveals the accelerated growth of iron-containing oxides such as hematite Fe2O3 and iron-chromite FeCr2O4, when the initial germination of the oxide layer contains the presence of a manganese-containing spinel compound (1000°C). When the initial growth shows the only chromia formation (800°C), hematite formation appears differed in time. Protection against corrosion is thus increased when the initial germination of manganese-containing spinel oxide is inhibited in the oxide scale.

Influence of Water Vapour on a Nickel-Based Alloy Oxidation

Water vapour has little effect on the oxidation rate and scale composition of a nickel-based SY 625 alloy oxidized between 900 and 1100°C. At 900 and 1000°C, the outer scale is composed of Cr2O3 and a continuous NbNi4 - Ni3Mo subscale is found at the oxide/alloy interface. At 1100°C the scale is composed of an outer chromia scale and an internal CrNbO4 subscale. The oxide scale morphology differs between dry and wet conditions. Under dry conditions the oxide scale appears to be compact and chromia pegs are observed at the internal interface. Under wet conditions, porosities are observed inside the scale. At 1100°C some scale spallation is observed under dry and wet conditions.

Effect of Phosphoric Acid Treatment on Isothermal High Temperature Oxidation Behaviour of AISI 304 Stainless Steel at 800°C

Phosphoric acid treatment is used as a way to improve the high temperature oxidation resistance of a chromia-forming AISI 304 steel. Chromia-forming steels are excellent candidates to resist to high temperature oxidizing atmospheres because of the formation of protective oxide scales. The oxide scale growth mechanisms are studied by exposing phosphoric acid-treated and untreated 304 steel samples to high temperature conditions in air. The analyses were carried out by means of thermogravimetry, and in situ X-ray diffraction (XRD). The experimental results show that the phosphoric acid treatment does not have a beneficial effect on cyclic high temperature oxidation (up to 70h of the oxidation test) of AISI 304 steel because of growth of a layer mainly formed by external cation diffusion which grows very quickly. The isothermal high temperature oxidation of this steel at 800°C in air shows a very fast initial iron oxidation towards the external interface, allowing to chromium element to be more available to the internal interface to form a continuous chromia layer, thus causing the establishment of a parabolic oxidation regime and leading to a beneficial reduction of the oxidation rate (after 70h of the oxidation test).

Influence of Water Vapour on Isothermal and Thermal Cyclic Oxidation Conditions of a Nickel-Based SY625 Steel at 1100°C

In isothermal oxidation condition, water vapour has little effect on the oxidation rate and scale composition of a nickel-based SY 625 alloy oxidized at 1100°C. The scale is composed of an outer Cr2O3 and an internal CrNbO4 scale. The oxide scale morphology differs between dry and wet conditions. Under dry conditions the oxide scale appears to be compact and chromia pegs are observed at the internal interface. Under wet conditions, porosities are observed spread inside the scale and the chromia grain size is smaller. At this temperature some scale spallation is observed under dry and wet conditions. Under cyclic oxidation conditions the oxide scale adherence is slightly improved in wet environment. The chromia scale is adherent during the 4 first oxidation cycles. In dry air, spallation occured after the first cycle. In dry and wet conditions, after the chromia scale spallation has started, NiO and NiCr2O4 form first. NiMoO4 forms later on the alloy surface during the cycling test. The best resistance of the alloy under thermal cycling conditions under wet conditions is related to the presence of a more plastic and adherent scale owing to a higher scale porosity and smaller chromia grain size compared to dry conditions.