Computational and conformational evaluation of FTase alternative substrates: insight into a novel enzyme binding pocket

Protein farnesyltransferase (FTase) is an important anticancer drug target. In an effort to develop isoprenoid diphosphate-based FTase inhibitors, striking variations have been observed in the ability of conservatively modified analogues to bind to the enzyme. For example, 2Z-GGPP is an alternative substrate with high binding affinity, while GGPP is not an alternative substrate. Using the availability of high-resolution FTase crystal structures, we have used pharmacophore and docking studies to elucidate a new binding pocket for isoprenoid analogues. The unique conformations between the first two isoprene units of 2Z-GGPP, but not GGPP, allows 2Z-GGPP to exploit this new binding pocket. The discovered conformation allows the molecule to adopt a reactive conformation while placing hydrophobic groups within the predominately hydrophobic binding pocket. This computational finding is supported by NMR studies on (13)C-labeled 2Z-farnesol, which confirm that the computationally predicted conformation is also favored in solution. These discoveries suggest that ligand conformational flexibility may be an important design consideration for the development of both inhibitors and alternative substrates of FTase.     

Analysis of Metribuzin and transformation products in soil by pressurized liquid extraction and liquid chromatographic-tandem mass spectrometry

A method developed for study of metribuzin degradation in soil is presented. LC-MS-MS and electrospray ionisation was used for analysis of metribuzin and the metabolites deaminometribuzin (DA), diketometribuzin (DK) and deaminodiketometribuzin (DADK). Soil samples were extracted by pressurized liquid extraction using methanol-water (75:25) at 60 degrees C. In general, recoveries were about 75% for metribuzin, DA and DADK and their detection limit in soil was 1.25 microg/kg. Lower sensitivity was observed for DK, with detection limit at 12.5 microg/kg and recovery about 50%.     

Null charts and naked singularities in spherically symmetric,homothetic spacetimes

We give a unified derivation of a null chart for all spherically symmetric, homothetic spacetimes. These spacetimes contain an interesting class of naked singularities which we are also able to elucidate. Much use is made of graphical representation; in particular a chart of the spacetimes based on their homothetic group motions is introduced. Dust spacetimes, and two homogeneous examples with non-zero pressure (flat Robertson-Walker and a Kantowski-Sachs example) are studied in detail. We show the horizon structure in the null atlas, in comoving coordinates, in terms of the areal radius and comoving time, and in the homothetic diagrams. The critical delay between comoving observers for the onset of nakedness is interpreted in terms of a decreasing mass concentration in the spirit of Thorne's hoop conjecture. We also give a simple criterion for the existence of apparent horizons isolating the various singularities, and study in detail how this criterion is circumvented in the naked examples. We conclude that this type of naked singularity is a consequence of the imposed homothetic symmetry, by showing it to be generally present and timelike in the homothetic group chart even when it is not visible at comoving infinity (before the onset of criticality). It is the delayed final collapse of initially distant observers in inhomogeneous spacetimes that causes the initial singularity to become visible at comoving infinity. We conclude that these examples do not present an obstacle to the Event Horizon Conjecture as summarized by W. Israel (1984). That is, one can formulate criteria for the formation of apparent horizons that do not imply that all singularities are necessarily so enclosed. It is still possible that all singularities stronger than homothetic are isolated by an apparent horizon, in the spirit of Tipler's conjecture.On leave from Department of Physics, Queen's University, Kingston, Ontario, Canada     

pH-sensitive paramagnetic liposomes for MRI: assessment of stability in blood

The pH-dependent stability of dipalmitoyl phosphatidyl ethanolamine/palmitic acid (DPPE/PA) liposomal GdDTPA-BMA was investigated in human blood and after exposure to selected blood components. Relaxometry, visual observations and cryo-transmission electron microscopy (cryo-TEM) were employed for the assessment of stability. The liposomes were stable in buffer at physiological pH and the T(1)-relaxivity (r(1)) of the system was significantly lowered compared to that of non-liposomal GdDTPA-BMA, which could be explained by an exchange limited relaxation process. Lowering the pH, however, gave a marked increase in r(1), due to liposome aggregation and subsequent leakage of GdDTPA-BMA. After a few minutes incubation in human blood the liposomes were destabilised and leaky at both high and low pH, and blood components likely to cause the instability were studied. Physiological level of Na(+) (150 mM) did not affect the relaxometric behavior of the liposomes at pH 7.4, but shifted the pH-r(1) profile laterally to higher pH-values compared to a level of 50 mM Na(+). Increased screening of the surface charges and, concomitantly, a lowering of the energy-barrier against aggregation is a plausible explanation for this phenomenon. In contrast, both Ca(2+) and Mg(2+) (physiological level, both 2 mM) caused massive aggregation of the liposomes and leakage of contents, and were therefore much more detrimental to the stability of the liposomes than a physiological level of Na(+). This could be due to the higher screening ability of divalent cations, but aggregation could also be induced through an inter-liposomal "bridging" effect. Physiological level of both Na(+) and Ca(2+) caused less leakage than for lower Na(+) concentration (50 mM Na(+) and 2 mM Ca(2+)), probably due to competition for the negative surface charges. Albumin also destabilised the liposomes, and it was shown to be due to an interaction between albumin and PA in the liposomal membrane.     

Determination of T1- and T2-relaxation times in the spleen of patients with splenomegaly

Twenty-nine patients with known splenomegaly and seven healthy volunteers were examined. The T1 and T2 relaxation times were read out from a region of interest centrally in the spleen. Even though different mean T1 and T2 relaxation times were found between the groups, the great scatter and the considerable overlap between the groups makes the contribution of relaxation time measurements to the differential diagnosis of splenomegaly of limited value.     

In vivo evaluation of the reproducibility of T1 and T2 measured in the brain of patients with multiple sclerosis.

The precision (reproducibility) of relaxation times derived from magnetic resonance images of patients with multiple sclerosis (MS) were investigated. Measurements of 10 MS patients were performed at 1.5 T on two occasions within 1 wk. T1 and T2 was measured using a partial saturation inversion recovery sequence (6 points) and a Carr-Purcell-Meiboom-Gill phase alternating-phase shift multiple spin-echo sequence with 32 echoes. Regions of interest (ROI) were placed both in apparently normal white matter and plaques. The precision (+/- 1.96 SD) and the confidence intervals for T1 and T2 for white matter and plaques were calculated. The precision of T1 for white matter and plaques was respectively +/- 94 msec and +/- 208 msec. The precision of T2 for white matter and plaques was respectively +/- 18 msec and +/- 26 msec. For all measurements the coefficient of variation was about 9%. Judging from our own study and others as well, a precision better than 10% for T1 and T2 would seem unrealistic at present.     

Combined experimental and theoretical study of the mechanism and enantioselectivity of palladium- catalyzed intermolecular Heck coupling

The asymmetric Heck reaction using P,N-ligands has been studied by a combination of theoretical and experimental methods. The reaction follows Halpern-style selectivity; that is, the major isomer is produced from the least favored form of the pre-insertion intermediate. The initially formed Ph-Pd(P,N) species prefers a geometry with the phenyl trans to N. However, the alternative form, with Ph trans to P, is much less stable but much more reactive. In the preferred transition state, the phenyl moiety is trans to P, but significant electron density has been transferred to the alkene carbon trans to N. The steric interactions in this transition state fully account for the enantioselectivity observed with the ligands studied. The calculations also predict relative reactivity and nonlinear mixing effects for the investigated ligands; these predictions are fully validated by experimental testing. Finally, the low conversion observed with some catalysts was found to be caused by inactivation due to weak binding of the ligand to Pd(0). Adding monodentate PPh3 alleviated the precipitation problem without deteriorating the enantioselectivity and led to one of the most effective catalytic systems to date.