Experimental evidence of Cr magnetic moments at low temperature in Cr2A(A=Al, Ge)C.

From x-ray magnetic circular dichroism experiments performed at low temperature on Cr2AlC and Cr2GeC thin films, it is evidenced that Cr atoms carry a net magnetic moment in these ternary phases. It is shown that the Cr magnetization of the Al-based compound nearly vanished at 100 K in agreement with what has been recently observed on bulk. X-ray linear dichroism measurements performed at various angles of incidence and temperatures clearly demonstrate the existence of a charge ordering along the c axis of the structure of Cr2AlC. All these experimental observations support, in part, theoretical calculations claiming that Cr dd correlations have to be considered to correctly describe the structure and properties of these Cr-based ternary phases.

Steric effect on the M site of nanolaminate compounds M(2)SnC (M = Ti, Zr, Hf and Nb).

In this paper we report calculations based on an all electron ab initio full-potential linearized augmented plane-wave method using the generalized gradient approximation within the density functional theory to determine the structures of Ti(2)SnC, Zr(2)SnC, Hf(2)SnC and Nb(2)SnC. The lattice constants obtained after geometry optimization are in good agreement with experimental data. It is observed from these results that there exists a steric effect on the M site. For M atoms with atomic radius (Zr, Hf) larger than that of tin, the polyhedra (octahedron and trigonal prism) constituting the unit cell are less distorted compared to those related to M atoms with atomic radius (Ti, Nb) similar to that of tin. The computed values for the bulk modulus of these ternary carbides are predicted to be about 69% of those of the corresponding binary carbides MC. The analysis of the projected local density of states shows that the major hybrids come from M (M = Ti, Zr, Hf, Nb) d and C p states.

HRTEM and EELS study of Y2O3/MgO thin films.

Y2O3 thin films deposited on (001)-MgO substrate have been investigated by high-resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy. Digital processing of the HRTEM images reveals the presence of grains with a crystallographic structure different from that of the rest of the film (Ia3). The spectrum imaging technique has been applied in vicinity of the Y2O3/MgO interface to get a better knowledge of the phases nucleated on the substrate surface. Fine structures of the O K-edge have been studied in detail; actually two kinds of spectra have been detected in the yttrium oxide thin film. These spectra have been compared to self-consistent full multiple scattering calculations (SC-FMS). One family of spectra has then been associated to the well-known Ia3 structure. The other family of spectra has been compared to calculations performed for the other known structures (such as hexagonal or monoclinic) of Y2O3 with a little success. We have finally compared these spectra to calculations performed with a particular atomic arrangement (octahedral) of Y and O atoms, which leads to a good match between experimental and calculated spectra. Our results emphasize the benefit of coupling several techniques such as HRTEM, EELS and SC-FMS for the determination of structures at the nanometric scale.

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450.

Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin. Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue. Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min(-1) P450(-1). Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man. Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.

Study of the local atomic strain field in a Zr-doped TiAl intermetallic alloy by EXAFS and ab initio FLAPW calculations.

The mechanical properties of gamma-TiAl-based intermetallic alloys are strongly influenced by Ti d-Ti d and Al p-Ti d hybridizations. These directional bonds supply good mechanical properties at high temperature but they are also associated with a low ductility at room temperature. Small amounts (about 3%) of additional elements can improve this behaviour noticeably and modify the mechanical properties. In the present case, the addition of zirconium in the gamma-TiAl-based alloys has been reported to increase their ductility at low temperature. Beyond the modifications of the directional bonds which come directly from the band structure, the different atomic volumes of the solute atoms induce local deformation fields which interact with the dislocations. We present ab initio calculations (FLAPW method) compared with an EXAFS study which the aim is to determine the site preference of zirconium in the TiAl lattice and the deformation fields around the solute atoms.

Inhibitory metabolite complex formation of methylenedioxymethamphetamine with rat and human cytochrome P450. Particular involvement of CYP 2D.

Methylenedioxymethamphetamine (MDMA or ecstasy) is a common recreational drug used at rave parties. Unfortunately, MDMA may have neurological effects and in some cases causes hepatotoxicity. MDMA binds to cytochrome P450 in rat and human hepatic microsomal preparations. Upon metabolic transformation of either the methylenedioxy or the methylamino function, it forms an inhibitory P450-metabolite complex. This inhibitory complex is formed predominantly with the P450 2D isozymes. This complex formation may account for the clinical toxicity observed upon ingestion of MDMA, particularly with other compounds normally metabolized by P450 2D6.

Detection of a nitric oxide synthase possibly involved in the regulation of the Rhodococcus sp R312 nitrile hydratase.

Crude homogenates from Rhodococcus sp 312 catalyze the conversion of L-arginine into L-citrulline and NO2-, the usual oxidation product of NO under aerobic conditions. They also catalyze the conversion of N omega-hydroxy-L-arginine (NOHA) into L-citrulline and NO2- with similar rates (10-15 and 100-150 nmol of product.min-1.(mg of protein)-1 respectively for the crude homogenate and for a fraction obtained from ammonium sulfate precipitation). L-citrulline formation is strongly inhibited by classical inhibitors of mammalian nitric oxide synthases (NOSs) such as N omega-methyl-L-arginine (NMA) and thio-L-citrulline (TC). Finally, the lack of inhibitory effects of EGTA, a classical inhibitor of constitutive mammalian NOSs, and the specific immunodetection of a 100 kD protein from Rhodococcus cytosol by an antibody raised against human inducible NOS, is in favor of the presence of a NOS similar to inducible mammalian NOSs in Rhodococcus sp 312. This NOS should be responsible for the NO-dependent inactivation of Rhodococcus Nitrile Hydratase (NHase) in the absence of light; it could regulate the activity of the latter enzyme.

Metabolism of tentoxin by hepatic cytochrome P-450 3A isozymes.

The interaction between rat and human liver cytochrome P-450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy. Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rat microsomes. Tentoxin exhibited a high affinity for P-450 3A (Ks approximately 10 microM). Similar results were observed with human P-450 isozymes expressed in yeast. Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation. Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin. The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision-activated dissociation modes. In addition to the major N-demethylated metabolite, other hydroxylated metabolites were formed. Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects.

Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand.

Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

Expression in yeast of three allelic cDNAs coding for human liver P-450 3A4. Different stabilities, binding properties and catalytic activities of the yeast-produced enzymes.

Three natural allelic cDNAs coding for P-450 3A4, the major form in human liver, namely NF25, NF10 and hPCN1, have been expressed in Saccharomyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO-reduced difference spectrum. Some catalytic activities and substrate binding properties of P-450 NF25 and P-450 hPCN1 in yeast microsomes have been compared; no striking difference was found, showing that the two point substitutions between their amino-acid sequences (Trp392 and Thr431 in P-450 NF25 are replaced by Val392 and Ile431 in P-450 hPCN1) have no significant effect on the functional properties of these two variants. By contrast, P-450 NF10, which differs from P-450 NF25 by a one-amino-acid deletion (Ile224 replacing Thr224-Val225), was produced as a denatured form, as revealed by an absorption maximum at 420 nm, and was not catalytically active. This suggests that the deletion prevents the correct folding of the protein. The results of this study show that P-450 NF25 and P-450 hPCN1 are two roughly equivalent, functionally active variants of P-450 3A4, but that P-450 NF10 is a defective, unstable gene product that could arise from an alternative mRNA splicing. This could contribute to the large variations reported for nifedipine oxidation, a typical P-450 3A4 activity, in human liver.

pH effects on the N-demethylation and formation of the cytochrome P-450 iron II nitrosoalkane complex for erythromycin derivatives.

The effects of pH on access to the cytochrome P-450 active site, N-demethylation and formation of the cytochrome P-450 Fe(II)-RNO metabolite complex for a series of erythromycin derivatives were examined. Studies were performed with dexamethasone-treated rat liver microsomes containing large amounts of cytochrome P-450 3A isozymes. In addition to factors such as hydrophobicity or hindrance around the dimethyl-amino function, the ionisation state of the N(CH3)2 group played an important role in the recognition and metabolism of the substrate by cytochrome P-450. Esterification of the desosamine in the beta position of the N(CH3)2 group leads to lower pKa values for the R--N+ H(CH3)2 <--> [R--N (CH3)2] + H+ equilibrium. At physiological pH, the amine group is mainly in the unprotonated form. Consequently, easier access to the protein active site and significant formation of cytochrome P-450 Fe(II)-RNO metabolite complex are observed for these derivatives. These results led us to interpret the formation of cytochrome P-450 Fe(II)-RNO metabolite complex as a series of multiple steps equilibria depending on the ionisation state of the N(CH3)2 group, the partition coefficient of the substrate between the microsomal layer and the aqueous media and a series of metabolic reactions leading partially to the final inhibitory nitrosoalkane-cytochrome P-450 Fe(II) complex.

Detection of human hepatitis anti-liver kidney microsomes (LKM2) autoantibodies on rat liver sections is predominantly due to reactivity with rat liver P-450 IIC11.

Anti-liver kidney microsomes (anti-LKM2) autoantibodies, appearing in patients treated with tienilic acid and suffering from hepatitis, react with proteins in rat liver sections. The nature of the rat proteins responsible for this recognition and detection of anti-LKM2 has been investigated. Immunoblot testing of the anti-LKM2 with liver microsomes from diversely treated rats and with purified rat liver cytochromes P450 (IA1, IA2, IIB1, IIB2, IIC6, IIC11 and IVA1) showed that these antibodies cross-reacted with cytochrome P450IIC11 and also with phenobarbital-induced cytochromes P450IIB1 and IIB2. Moreover, metabolic activation of tienilic acid and of a tienilic acid isomer by untreated rat liver microsomes was partially inhibited by anti-LKM2. On the other hand, monospecific polyclonal anti-rat P450IIC11 antibodies cross-reacted with human microsomal cytochromes P450 and recognized the same cytochromes P450 as anti-LKM2. This antibody also gave an immunofluorescence pattern on rat and mouse liver and kidney sections very similar to anti-LKM2. The data presented here show that anti-LKM2 recognize epitopes shared by rat P450 IIC11, and a human P450 of the family IIC. All the results indicate rat P450 IIC11, the major isoenzyme present in normal adult male rat liver, as the main antigen recognized by human anti-LKM2 autoantibodies; this is the basis of the immunofluorescence test for detection of these antibodies.

Vitamin E derivatives as new potent inhibitors of microsomal lipid peroxidation.

Several synthetic Vitamin E derivatives are strong inhibitors of lipid peroxidation induced in rat liver microsomes either chemically by ferrous ions and ascorbate or enzymatically by NADPH and carbon tetrachloride. The relative activities of these inhibitors are consistent with their intrinsic antioxidant properties, as peroxyl radicals scavengers. Among them, a 3,4-dihydro-6-hydroxy-2H-1-naphtopyran with IC50 around 0.08 microM is one of the most potent yet known inhibitor of lipid peroxidation.

Microsomal lipid peroxidation and oxy-radicals formation are induced by insoluble iron-containing minerals.

Lipid peroxidation, measured by malondialdehyde formation is induced in rat liver microsomes by insoluble iron-containing minerals (pyrite, magnetite, nemalite and an iron ore, minette de Lorraine) which are generally found either in iron mines or as contaminants of asbestos fibers. In spin-trapping studies using DMPO as a spin trap those minerals are also found to catalyze the oxidation of formate to carboxylate radicals by oxygen, via the formation of hydroxyl radicals. The two processes are mainly due to the presence of redox active iron at the surface of the solid particles and thus are greatly inhibited by desferrioxamine, a strong iron chelator. However, these reactions are not correlated.

In vivo effects of immunostimulating lipopeptides on mouse liver microsomal cytochromes P-450 and on paracetamol-induced toxicity.

Immunomodulating lipopeptides lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2-Gly (RP 44.102) and lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2 (RP 56.142) were found to protect mice against the hepatotoxicity of paracetamol, which is due to cytochrome P-450 dependent formation of toxic metabolites and radicals. In fact they decreased the amount of hepatic microsomal cytochrome P-450, and the level of CCl4-induced lipid peroxidation. In contrast lauroyl-L-Ala-gamma-D-Glu-DD-A2pmNH2 (RP 53.204), which only differs by the configuration of the two chiral carbons of A2pm (diaminopimelic acid) and is not an immunomodulating agent, failed to protect against poisoning by paracetamol and had no effect on the level of hepatic cytochrome P-450 or the microsomal CCl4-induced lipid peroxidation. This provides a clear connection between the immunostimulating properties of a compound and its effects on xenobiotic biotransformations.

The stimulatory effects of asbestos on NADPH-dependent lipid peroxidation in rat liver microsomes.

Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive.

Drug interactions with macrolide antibiotics: specificity of pseudo-suicide inhibition and induction of cytochrome P-450.

Macrolide antibiotics like Erythromycin and Tri-acetyl oleandomycin (TAO) are metabolized to nitrosoderivatives which cause inactivation of Cytochrome P-450 by forming stable complex with the Iron of the hemoporphyrin. Several derivatives of erythromycin having lost their cladinose moiety are stronger inducer of liver cytochrome P-450 itself. The major form of cytochrome P-450 induced by all these macrolides in rat liver electrophoretically and immunologically indistinguishable from the major form induced by pregnenolone 16 alpha carbonitrile (PCN). This form is particularly able to metabolize macrolide and to lead to the corresponding 456 nm absorbing cytochrome P-450 complexes in vivo and in vitro.