Philippe Gallice

Binding of p-cresylsulfate and p-cresol to human serum albumin studied by microcalorimetry.

p-Cresylsulfate, a metabolite of p-cresol, is reported as prototypic protein-bound uremic toxin, inefficiently removed by haemodialysis. The binding between p-cresylsulfate or p-cresol and human serum albumin was studied using microcalorimetry. The results confirm that the two molecules are protein-bound. However, the affinity of p-cresylsulfate and p-cresol toward human serum albumin is moderate at 25 degrees C and becomes relatively weak at physiological temperature, 37 degrees C. The binding principally involves van der Waals type interactions, and the binding sites of the two molecules are the same or very close. The low fraction of bound toxin (13-20%) appears to be insufficient to link strong binding to poor removal of this toxin by hemodialysis.

Influence of the Length of Imogolite-Like Nanotubes on Their Cytotoxicity and Genotoxicity toward Human Dermal Cells

Physical-chemical parameters such as purity, structure, chemistry, length, and aspect ratio of nanoparticles (NPs) are linked to their toxicity. Here, synthetic imogolite-like nanotubes with a set chemical composition but various sizes and shapes were used as models to investigate the influence of these physical parameters on the cyto- and genotoxicity and cellular uptake of NPs. The NPs were characterized using X-ray diffraction (XRD), small angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Imogolite precursors (PR, ca. 5 nm curved platelets), as well as short tubes (ST, ca. 6 nm) and long tubes (LT, ca. 50 nm), remained stable in the cell culture medium. Internalization into human fibroblasts was observed only for the small particles PR and ST. None of the tested particles induced a significant cytotoxicity up to a concentration of 10(-1) mg·mL(-1). However, small sized NPs (PR and ST) were found to be genotoxic at very low concentration 10(-6) mg·mL(-1), while LT particles exhibited a weak genotoxicity. Our results indicate that small size NPs (PR, ST) were able to induce primary lesions of DNA at very low concentrations and that this DNA damage was exclusively induced by oxidative stress. The higher aspect ratio LT particles exhibited a weaker genotoxicity, where oxidative stress is a minor factor, and the likely involvement of other mechanisms. Moreover, a relationship among cell uptake, particle aspect ratio, and DNA damage of NPs was observed.

NMR and MD Investigations of Human Galectin-1/Oligosaccharide Complexes

The specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related beta-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1.

Modulation defect of sodium pump evidenced in diabetic patients by a microcalorimetric study

Sodium pump activity of intact erythrocytes in their own plasma was measured by microcalorimetry in 41 healthy subjects and 35 insulin-dependent diabetic patients. Results show that modulation of the sodium pump is altered in diabetic patients. Addition of insulin increases functioning of the Na(+)-K+ pump in controls but has no effect in diabetic patients. These subjects show a slower response of the Na(+)-K+ pump to the inhibitory effect of ouabain. Cross-incubation experiments suggest that these findings may be explained by the existence of a plasmatic factor that impairs the modulation of the sodium pump in diabetic patients.

SIMULTANEOUS GC/MS ANALYSIS OF POLYCYCLIC AROMATIC HYDROCARBONS AND THEIR NITRATED DERIVATIVES IN ATMOSPHERIC PARTICULATE MATTER FROM WORKPLACES

Abstract PAH are routinely analyzed using HPLC/FD. This technique is unsuitable for analyzing NPAH. This study aims at developing a reliable method, using GC/MS, and applying this technique to actual samples from small volumes of atmospheric particulate matter from workplaces. Mixtures of PAH and NPAH were separated by GC/MS and detected by electronic impact (EI) or negative ion chemical ionization (NICI). Analyses on twelve actual samples were thus carried out by sampling a small volume of atmosphere (≈0.5 m 3 ) from five different industrial workplaces. Samples displayed wide differences from one industrial workplace to another, and this can be explained by the specific methods applied. The PAH and NPAH concentrations also varied with time in the same industrial workplace. NPAH concentrations were not correlated with PAH concentrations, underscoring the complex chemical mechanisms involved in NPAH formation. PAH and NPAH formation appeared to be dependent on both industrial activities and uncontrolled physicochemical conditions.

A non ouabain-like inhibitor of the sodium pump in uremic plasma ultrafiltrates and urine from healthy subjects.

A non ouabain-like inhibitor of the sodium pump was separated from uremic plasma ultrafiltrates and normal urine. Under the same chromatographic conditions (C18 column and a gradient of acetonitrile as eluant), ouabain was eluted in a fraction different from the inhibitor. Affinity chromatography based on the formation of a complex between Na,K-ATPase and the inhibitor achieved the differentiation ouabain. Without magnesium and sodium phosphate, ouabain could not bind to enzyme whereas the inhibitor did. A study of Na,K-ATPase enzyme kinetics showed the inhibitor was not competitive for K+, which further differentiates it from ouabain. It was uncompetitive for ATP and seemed competitive for Na+. These results indicate that the inhibitor acts inside the cell, unlike ouabain, and thus its action mechanism appears to be original.

Sodium pump and Na+/H+ activities in uremic erythrocytes. A microcalorimetric and pH-metric study.

The sodium pump and Na+/H+ antiport activities in red blood cells from uremic hemodialyzed patients were measured concomitantly. The patients selected (n = 35) were normotensive and free of intercurrent illness known to affect Na transport. The Na pump activity of intact red blood cells in suspension in their own plasma was measured by flow microcalorimetry. The Na+/H+ antiport activity of the erythrocytes from the same patients was determined by a titrimetric technique. The mean global value of the sodium pump was lower in uremics than in controls (13.3 +/- 0.6 vs. 11.3 +/- 0.8 mW/l cells, P < 0.05). The Na+/H+ antiport maximal activity was decreased in uremics (2.9 +/- 0.3 vs. 4.6 +/- 0.5 mmol H+/l cells/h, P < 0.05). Our results thus confirm that uremia per se can affect sodium transport. Moreover it has been shown that a decrease in Na+/H+ antiport activity is able to counteract an impairment of sodium pump. The decrease found in this study could thus explain, at least in part, the absence of hypertension in the patients studied despite their decreased sodium pump activity.

Ascorbic acid-2-0-β-glucuronide, a new metabolite of vitamin C identified in human urine and uremic plasma

A new metabolite of ascorbic acid has been isolated by a multi-step chromatographic procedure both from normal human urine and uremic plasma. Nuclear Magnetic Resonance studies, and chemical and enzymic analyses indicated that the compound is a conjugated structure consisting of equimolar ascorbic and beta-D-glucuronic acids. We determined the pKa value of the ascorbic acid moiety of the compound on the basis of variations of ultraviolet absorbances as a function of pH. Results showed that glucuronic acid is coupled to the 2-position of ascorbic acid.

Phosphorus-31 and water proton relaxation in living erythrocytes. Application to uremia

We measured the spin-lattice and spin-spin relaxation times (T1 and T2, respectively) and the nuclear Overhauser effect (NOE) of 31P nuclei of 2,3-diphosphoglycerate (2,3-DPG) in living erythrocytes. The relaxation of water protons was also studied. Phosphorus relaxation is pH-dependent due to a modification of the binding of 2,3-DPG to hemoglobin. We compared the results obtained with normal and uremic erythrocytes. In uremic erythrocytes the 31P relaxation rates are increased, but the intraerythrocytic pH variation in uremic erythrocytes cannot itself explain this increase. A possible role of dialysable substances may explain the increased relaxation rate.

Scaling up in isolation of medium-size uraemic toxins

Plasmatic accumulation of uraemic toxins in the middle molecular mass range has been reported to be associated with several pathologies observed in uraemic patients. The very low concentration of these toxins in uraemic body fluids makes classical chromatography techniques inadequate in isolating sufficient amounts of these endogenous substances, thus precluding their identification. A scaling up of gel permeation and ion-exchange chromatographies was therefore developed. This considerably increased the amount of uraemic toxins isolated, thus allowing the study of their chemical nature and facilitating understanding of their biological activities.