Defects in Stratum Corneum Desquamation Are the Predominant Effect of Impaired ABCA12 Function in a Novel Mouse Model of Harlequin Ichthyosis.

Harlequin Ichthyosis is a severe skin disease caused by mutations in the human gene encoding ABCA12. Here, we characterize a novel mutation in intron 29 of the mouse Abca12 gene that leads to the loss of a 5' splice donor site and truncation of the Abca12 RNA transcript. Homozygous mutants of this smooth skin or smsk allele die perinatally with shiny translucent skin, typical of animal models of Harlequin Ichthyosis. Characterization of smsk mutant skin showed that the delivery of glucosylceramides and CORNEODESMOSIN was defective, while ultrastructural analysis revealed abnormal lamellar bodies and the absence of lipid lamellae in smsk epidermis. Unexpectedly, mutant stratum corneum remained intact when subjected to harsh chemical dissociation procedures. Moreover, both KALLIKREIN 5 and -7 were drastically decreased, with retention of desmoplakin in mutant SC. In cultured wild type keratinocytes, both KALLIKREIN 5 and -7 colocalized with ceramide metabolites following calcium-induced differentiation. Reducing the intracellular levels of glucosylceramide with a glucosylceramide synthase inhibitor resulted in decreased secretion of KALLIKREIN proteases by wild type keratinocytes, but not by smsk mutant keratinocytes. Together, these findings suggest an essential role for ABCA12 in transferring not only lipids, which are required for the formation of multilamellar structures in the stratum corneum, but also proteolytic enzymes that are required for normal desquamation. Smsk mutant mice recapitulate many of the pathological features of HI and can be used to explore novel topical therapies against a potentially lethal and debilitating neonatal disease

Unusual asymmetry of methyl /sup 2/H EFG in thymine: a solid state deuterium NMR and ab initio MO study

Solid-state /sup 2/H NMR spectroscopy is a powerful method of studying molecular motion in the solid state. The power of this technique derives from the facts that quadrupole coupling is the dominant interaction and the electric field gradient (EFG) tensor is usually axially symmetric for a deuteron bonded to carbon, with the unique axis along the C-D bond. In the case of a methyl group, rapid 3-fold rotation yields an averaged EFG whose symmetry axis is along the rotation axis, and the corresponding powder pattern is axially symmetric (eta less than or equal to 0.01) with a quadrupole splitting of ca. 40 kHz. They are interested in obtaining spectra of thymidine and ribothymidine, deuteriated at the methyl positions, to study motion of nucleotide bases in DNA and tRNA, respectively. In their initial work they are studying the dynamics of the model compound, thymine-methyl-d/sub 3/. Inversion-recovery /sup 2/H NMR spectra of this compound clearly showed the T/sub 1/ anisotropy predicted for 3-fold methyl jumps. The linear Arrhenius plot of correlation time, tau/sub c/, against 1/T (where tau/sub c/ = tau/sub 0/ exp(E/RT)) yielded an apparent activation energy of 6.9 kJ/mol and a preexponential factor, tau/sub 0/, of 2 x 10/sup -13/ s. These results show that the /sup 2/H spin-lattice relaxation is determined by the 3-fold methyl motion. However, this motion does not account for the asymmetry in the observed line shape

Improved detection of long-range residual dipolar couplings in weakly aligned samples by Lee-Goldburg decoupling of homonuclear dipolar truncation

Homonuclear (1)H residual dipolar couplings (RDCs) truncate the evolution of transverse (1)H magnetization of weakly aligned molecules in high-resolution NMR experiments. This leads to losses in sensitivity or resolution in experiments that require extended (1)H evolution times. Lee-Goldburg decoupling schemes have been shown to remove the effects of homonuclear dipolar couplings, while preserving chemical shift evolution in a number of solid-state NMR applications. Here, it is shown that the Lee-Goldburg sequence can be effectively incorporated into INEPT- or HMQC-type transfer schemes in liquid state weak alignment experiments in order to increase the efficiency of the magnetization transfer. The method is applied to the sensitive detection of (1)H(N)-(13)C long-range RDCs in a three-dimensional HCN experiment. As compared to a conventional HCN experiment, an average sensitivity increase by a factor of 2.4 is obtained for a sample of weakly aligned protein G. This makes it possible to detect 170 long-range (1)H(N)-(13)C RDCs for distances up to 4.9 angstroms