Carbide and nitride phase characterization in a transition metal carbo-nitride using x-ray spectroscopy and atom probe tomography.

A multi-phase hafnium carbo-nitride was investigated by various analytical methods. Incomplete homogenization between mixed HfC-HfN starting powders subjected to hot isostatic pressing resulted in both carbon-rich and nitrogen-rich phases. The compositions of these two phases were quantified in detail by wavelength dispersive spectroscopy and atom probe tomography, with the atom probe tips having either a small or a large shank angle geometry. For each of the two phases, an agreement of the compositions obtained by wavelength dispersive spectroscopy and atom probe tomography was found. However, the quality of the mass spectrum and hit multiplicity (single hits) were generally higher for the carbon-rich as compared to the nitrogen-rich carbo-nitride. Though the atom probe tip geometry does not appear to influence the composition, the mass resolving power did improve with the larger shank angle geometry while the hit multiplicity deteriorated slightly. Finally, our results demonstrate that hafnium carbide requires less thermal assistance to field evaporate than hafnium nitride.

Complex evaporation behavior of a transition metal carbo-nitride (Hf(C,N)) studied by atom probe tomography.

The use of pulsed lasers in atom probe tomography has enabled the analysis of lower conductivity materials such as hafnium carbo-nitrides. The variability of experimental parameters can have a profound effect on field evaporation behavior, data quality and compositional accuracy. This is especially challenging for materials such as hafnium carbo-nitride, where a mixture of covalent, ionic and metallic bonding types is present. Here we study the influence of laser pulse energy on how the field evaporation evolves in a hafnium carbo-nitride and how that impacts data quality and compositional accuracy. Changing the laser pulse energy, while keeping other parameters constant, alters the resulting composition. A gain in Hf concentration is observed for higher laser pulse energies while at the same time the N concentration decreases. At lower laser pulse energies, the obtained composition is in good agreement with the reference bulk composition of the material. Furthermore, our results demonstrate that assessing the quality of an APT experiment or dataset merely based on commonly used metrics such as quality of mass spectrum, hit distribution on the detector, hit multiplicity and mass resolving power, can be misleading and is not enough to ensure the most accurate compositional data. Moreover, it is shown that the complex evaporation behavior of transition metal carbo-nitrides and potential ion loss mechanisms are not well enough understood yet and further work is required to fully comprehend these complex behaviors in these types of ceramics.

Brain fat embolism. A report of two cases and a brief review of neuroimaging findings.

Fat embolism syndrome [FES] is an uncommon but serious complication of traumatic injures, which can follow a wide range of other surgical and medical conditions and can manifest with a collection of respiratory, hematological, neurological and cutaneous symptoms. FES should be suspected in all cases of traumatic injures with altered mental status following a period of normal neurological function, especially after orthopedic fixation of long bone fractures. Neurological symptoms must not be related to the initial trauma. The objective of this study was to describe the characteristics of FES and to correlate the neuroradiological findings with the clinical symptoms and the outcome of two cases of cerebral FES which occurred in two young men after bone fractures of the extremities without cranial traumatism. Both patients were studied by brain computed tomography (CT) and by brain magnetic resonance (MR). While Cerebral CT was inadequate to the diagnosis, Cerebral MR, performed 48 hours after injury, showed several focal areas of pathological signal in the white matter of the subcortical, periventricular and centrum semiovale regions, as well as in the basal ganglia and cerebellum. The neuroradiological findings of the two patients were compared with their clinical symptoms and outcome. The patient with the worst prognosis showed more lesions on MR and a restricted diffusion on DWI-MR, due to cytotoxic edema, whereas the patient with the better outcome showed lesions due to vasogenetic edema without any restricted diffusion. Cerebral MR and DW-MR are sensitive indicators for the early diagnosis of FES and can give a vast amount of information on the prognosis and future outcome.

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells.

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.

The membrane anchor of microsomal epoxide hydrolase from human, rat, and rabbit displays an unexpected membrane topology.

The microsomal epoxide hydrolase (mEH) and cytochrome P450s catalyze the sequential formation of carcinogenic metabolites. According to one algorithm for predicting the membrane topology of proteins, the human, the rabbit, and the rat mEH should adopt a type II topology. The type II topology is also predicted by a recently established neuronal network which is trained to recognize signal peptides with very high accuracy. In contrast to these predictions we find, based on N-glycosylation analysis in a cell-free and in a cellular system, that the membrane anchor of human, rat, and rabbit mEH displays a type I topology. This result is correctly predicted by the positive inside rule in which negatively charged residues, the distribution of which differs in the mEH membrane anchor of these species, have only a modulating role for the membrane topology of proteins. However, our results demonstrate that this role is not strong enough to force the mEHs into a type II topology, not even in the case of the rabbit mEH, in which the only positively charged residue in the C-terminal part of the topogenic sequence is flanked by five negatively charged residues.

The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology.

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein.

The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.