High resolution 3-T MR imaging in the evaluation of the facial nerve course.

OBJECTIVES: To assess the value of 3-Tesla (3-T) MR imaging (MRI) in the evaluation of the course of the intracranial and extra-cranial tracts of the facial nerve. PATIENTS AND METHODS: 83 patients were studied by MRI in order to detect the course of facial nerve; a total of 166 facial nerves were examined. T2-weighted 3D Fast imaging employing steady-state acquisition (FIESTA) and T1-weighted Fast spoiled gradient recalled echo (fast SPRG) sequences were used. Two radiologists (reader A and B), independently, evaluated the course of the tracts of the facial nerve according to a qualitative scale (excellent, good, fair, poor). The Intraclass Correlation Coefficient (ICC) and Pearson correlation coefficient were used to assess the intra-observer and interobserver variability in the nerve course evaluation. RESULTS: Reader A evaluated 35 facial nerves as excellent, 94 as good, 33 as fair and 4 as poor. Reader B rated 31 facial nerves excellent, 89 good, 43 fair and 3 poor. The intraobserver variability was ICC = 0.919 in reader A and ICC = 0.842 in reader B. The interobserver variability (Pearson correlation coefficient) was 0.713 (p ≤ 0.01). CONCLUSIONS: According to the preliminary results of our study the use of 3-T MRI with FIESTA and fast SPGR sequences may allow the study of the course of the facial nerve and its branches. The knowledge of the course and of the anatomic relationships of these nerve bundles with surrounding structures, as well as of the anatomical variants, provide useful informations for a prompt neurosurgery and maxillofacial surgical planning.

Structure of an N-terminally truncated Nudix hydrolase DR2204 from Deinococcus radiodurans.

Nudix pyrophosphatases are a well represented protein family in the Deinococcus radiodurans genome. These hydrolases, which are known to be enzymatically active towards nucleoside diphosphate derivatives, play a role in cleansing the cell pool of potentially deleterious damage products. Here, the structure of DR2204, the only ADP-ribose pyrophosphatase in the D. radiodurans genome that is known to be active towards flavin adenosine dinucleotide (FAD), is presented at 2.0 angstrom resolution.

Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.

Mycobacterium tuberculosis TMP kinase (TMPK(Mtub)) represents a promising target for developing drugs against tuberculosis because the configuration of its active site is unique in the TMPK family. To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography. Six new structures of TMPK(Mtub) were solved at a resolution better than 2.3A, including the first structure of an apo-TMPK, obtained by triggering catalysis in a crystal of a TMPK(Mtub)-TMP complex, which resulted in the release of the TDP product. A series of snapshots along the reaction pathway is obtained, revealing the closure of the active site in going from an empty to a fully occupied state, suggestive of an induced-fit mechanism typical of NMPKs. However, in TMPK(Mtub) the LID closure couples to the binding with an unusual location for a magnesium ion coordinating TMP in the active site. Our data suggest strongly that this ion is required for catalysis, acting as a clamp, possibly in concert with Arg95, to neutralise electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions. The 3'-hydroxyl moiety of TMP, critical to metal stabilisation, appears to be a target of choice for the design of potent inhibitors. On the other hand, the usual NTP-bound magnesium is not seen in our structures and Arg14, a P-loop residue unique to TMPK(Mtub), may take over its role. Therefore, TMPK(Mtub) seems to have swapped the use of a metal ion as compared with e.g. human TMPK. Finally, TTP was observed in crystals of TMPK(Mtub), locked by Arg14, thus providing a structural explanation for the observed inhibitory effect of TTP putatively involved in a mechanism of feedback regulation of the enzymatic activity.

Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug.

Clonidine, clinically used in the treatment of hypertension, is a central alpha(2)-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity. However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K(i) for clonidine binding to NOS-I increased (from [7. 9 +/- 0.4] x 10(-5) M to [8.0 +/- 0.4] x 10(-3) M) as BH(4) concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees. In addition, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.

Cys25-nitrosylation inactivates papain.

Nitric oxide (NO) may modulate the catalytic activity of cysteine proteases. In the present study, the inhibitory effect of NO, released by the NO-donors (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide and nitroprusside, on papain action is reported. Papain inactivation via NO-mediated nitrosylation of the Cys25 catalytic residue represents a molecular model for cysteine protease inhibition.