Mutations in GDI1 and X-linked non-specific mental retardation.

Mental retardation (MR) is a complex phenotype characterized by suboptimal functioning of the central nervous system (CNS). It is estimated that from 1 to 3% of the general population is affected with MR. MR or "intellectual disability" can be caused by genetic as well as environmental causes that act on the development and functioning of the CNS prenatally, perinatally or postnatally. Genetic causes of MR include chromosome aneusomies, chromosome structural abnormalities, genomic disorders and monogenic diseases. Amongst children, acute MR (QI < 50) is estimated at 0.4% and faint MR is about 2.5-3%. To determine the etiology of the MR, many diagnostic studies have been conducted and they show that MR is very heterogeneous and its etiology is not yet known in 20-50% of the group of patients with severe MR. This percentage increases up to 75-80% in the group of individuals with mild or "borderline" forms of MR. In light of the literature results, we tried to carry out a screening of 41 subjects with nonspecific MR for the detection of mutations in the gene GDI1 using the DHPLC methodology. This technique has the following advantages: low cost, high sensitivity (> 95%), and it can be done quickly. We have found 3 nucleotide (nt) substitutions: an intronic polymorphism at nt 107877 A --> C, a polymorphism in exon 3 at nt 109259 T --> C (Asn73Asn), and an intronic polymorphism at nt 110314 G --> C. The mutations in this gene are common and do not seem to influence the gene expression so as to cause a change in phenotype. These results therefore do not encourage the research of a diagnostic protocol designed for mutational analysis of the GDI1 human gene as the only responsible factor for a complex disease as Mental Retardation X-linked (MRX).

MDR-related properties of K 562 cells grown in two different culture media.

Using a synthetic substitute, Ultroser HY, for fetal bovine serum to supplement a classical RPMI 1640 culture medium produced changes in the properties of sensitive and of multidrug-resistant K 562 cells. Though no morphological changes were found, a statistically significant decrease in doubling-time was noted. Plasma membranes were more rigid, as reflected by an increase in the order parameter values. Adriamycin cytotoxicity was decreased, as shown by an increase in IC 50 values. The THP-adriamycin uptake, monitored by fluorimetry, was diminished even when the revertant agent verapamil was added. Moreover, the apparent number of Pgp 170 molecules per cell was lower for resistant cells grown with Ultroser HY. Thus Pgp 170 was not involved in the MDR increase induced by Ultroser HY. In conclusion, it must be kept in mind that environmental factors such as media chemical composition influence the MDR phenomenon and that environmental factors may also influence the MDR phenomenon in clinical situations.

Effects of benfluorex metabolites on membrane fluidity and insulin-related processes.

As little work has dealt with the antihyperglycemic property of benfluorex at the hepatocyte level, we studied the effects of its main metabolites, S422 and S1475, on membrane fluidity and on insulin binding, internalization and action in healthy rat hepatocytes. Both metabolites were effective fluidizing agents. Neither one affected insulin binding. Only S422 favored the bound insulin-receptor internalization process. The metabolites produced no change in basal alpha-aminoisobutyric acid uptake. Only S422 promoted the insulin-stimulated alpha-aminoisobutyric acid uptake in a dose-dependent way. Therefore, our study demonstrated that: (i) the effects of S422 on insulin-related processes in isolated hepatocytes were direct, specific and not due to any membrane fluidizing mechanism; (ii) S422 improved hepatocyte response to insulin at a post-binding level. These results in vitro give an additional explanation, at the cellular level, of the benefit of benfluorex treatment for non insulin-dependent diabetes patients.

In vitro influence of benfluorex and its main metabolites on rat liver microsomal membrane properties.

Benfluorex and its three main metabolites at 30 microM have been shown to inhibit Acyl CoA cholesterol acyl transferase activity in rat liver microsome preparations and to fluidize these membranes, as reflected by a decrease in the lipid order parameter. When drug concentrations were higher (60-200 microM), the compounds differed in their enzymatic inhibition properties but retained the same fluidizing effects. Only the parent compound had a dose-dependent inhibiting effect. These results are discussed with regard to the chemical properties of compounds, in particular their electric charges and their lipophilic characters.

Variations in fluorescence and enzymic properties of bovine dihydrofolate reductase.NADPH complex during the slow conformational change induced by coenzyme binding.

When NADPH was added in large excess to bovine dihydrofolate reductase (H2folate reductase), there was a slow isomerization process between two conformers of the binary complex (B1<-->B2), as shown by changes in the fluorescence properties. Thus, we monitored the time dependence of (a) the quenching of protein intrinsic fluorescence intensity, (b) the polarization state of the fluorescence light emitted by NADPH.H2folate reductase complexes and (c), from a more biological point of view, the enzymic activity of binary complex solutions. The kinetics for these three processes were in good agreement using the same temperature conditions. Furthermore, fluorescence studies provided information on the NADPH environment in the binary complex. As soon as NADPH bound to H2folate reductase, light emitted by the invariant Trp24 residue located within the coenzyme-binding site was quenched by an energy-transfer process. Moreover, Trp57 and/or Trp113 emissions were partially quenched. The subsequent NADPH-bound protein conformational change caused an additional quenching, probably of Trp57 and/or Trp113 emissions. Thus, NADPH.H2folate reductase conformation was modified but no change was observed at the coenzyme-binding site, at least in our fluorescence study. These results were confirmed by polarization measurements. The conformational change, as well as the instantaneous NADPH binding, resulted in a more rigid form of the protein, as shown by an increase in steady-state anisotropy values. Finally, the isomerization process led to a more active enzymic form.

Influence of cetiedil on erythrocyte membrane microviscosity and acetylcholinesterase activity.

Since one of the cellular targets of cetiedil, a vaso-erythroactive drug, is likely to be the erythrocyte membrane, we have studied the influence of this drug on erythrocyte membrane microviscosity and acetylcholinesterase activity. No effect was evidenced on microviscosity, as measured by fluorescence polarization of light emitted by DPH or TMA-DPH labelling of the lipid bilayer. Cetiedil, however, did lower acetylcholinesterase activity, but it did not directly inhibit this enzyme activity. It can therefore be considered as an amphiphilic drug that perturbs membrane properties without affecting the physical state of the erythrocyte membrane.

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.

Lipid diet and enterocyte microsomal membrane fluidity in rats.

Rats were fed on diets more or less enriched with n-3 and n-6 unsaturated fatty acids, before removal of the small intestine. The global protein, cholesterol and phospholipid contents of enterocyte microsomes were measured. Fatty acids of the total lipid extracts were determined. Acyl coenzyme A: cholesterol acyl transferase (ACAT) was chosen as the enzyme whose activity reflects metabolic changes induced by lipid diets. Fluorescence measurements using diphenylhexatriene as the membrane probe were performed. As dietary fat may change the fatty acid composition of membranes, the order parameter S calculated from fluorescence measurements was studied with regard to dietary fatty acid composition. The S values, distributed over a large range, were not different between rat groups. They were positively correlated with the ratios of cholesterol and proteins to phospholipids and the molar percentage of saturated fatty acids. ACAT activity was negatively correlated with S. Variations in S values among rats, whatever the diet, could in part be attributed to individual factors.

Influence of the guanine nucleotide phosphorylation state and of Mg2+ ions on the interaction of vinzolidine/tubulin 6 S: a fluorescence quenching study.

The binding of the new vincaalkaloid vinzolidine to tubulin 6 S was investigated by using fluorescence quenching methods. The value of the apparent equilibrium binding constant was found to depend on the phosphorylation state of the guanine nucleotide bound to the tubulin exchangeable site (E-site), with Ka values of 4.9 X 10(4) and 8.19 X 10(4) M-1 for GTP- and GDP-tubulin, respectively. The effect of Mg2+ ions on this binding was more important on GTP-tubulin than on GDP-tubulin, and might be related to the existence of Mg2+ site(s) independent of the nucleotide.

Fluorescence study of human beta 2 microglobulin.

A fluorescence study human beta 2 microglobulin showed the existence of two types of Trp residues, one quite exposed to the solvent, the other buried in a hydrophobic environment. The change in excitation wavelength made obvious the existence of a Tyr to Trp energy transfer mechanism. Treatment by urea or guanidine chlorhydrate brought about quite different results. With the former denaturing agent, some Trp residues remained buried; with the latter, the protein was completely unfolded, as proved by iodide quenching. pH variations could not unfold beta 2m enough to convert all Trp residues to exposed ones. When heated, beta 2m supported a transition that began at 50 degrees (melting temperature 63 degrees) and was not reversible. All these results suggest a rather compact conformation as in a globular protein.

Interaction of methotrexate metabolites with beef liver dihydrofolate reductase-I. Binary complex study.

In order to explain the difference of inhibition of dihydrofolate reductase (DHFR) by methotrexate (MTX) and its metabolites 7-hydroxymethotrexate [7OH (MTX)] and polyglutamate derivatives [MTX (G1) and MTX (G2)], direct determinations of binding parameters to beef liver DHFR were performed. Association constants are calculated by fluorescence titrations and thermodynamic parameters by microcalorimetric measurements. The parameters of interaction are nearly identical for MTX and polyglutamate derivatives but are different for MTX and 7OH (MTX). For this last derivative electrostatic forces are less predominant and a larger modification of its conformation appears in the enzyme during the complex formation.