Interactions of the alpha-spectrin N-terminal region with beta-spectrin. Implications for the spectrin tetramerization reaction

Spectrin of the erythrocyte membrane skeleton is composed of alpha- and beta-spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of alpha-spectrin (Nalpha region) bundles with another fractional domain in the carboxyl-terminal region of beta-spectrin (Cbeta region) to yield a triple alpha-helical bundle and that this helical bundling is largely responsible for tetramer formation. However, there are certain objections to assigning a preeminent role to this helical bundling in the tetramerization reactions. We prepared several recombinant peptides of alpha-spectrin fragments spanning only the Nalpha region (lacking the dimer nucleation site) and quantitatively studied their interaction with beta-spectrin. We found that a majority of the interactions were localized, as expected, in the Nalpha-helix C region but that there was also some contribution from the nonhomologous region. More importantly, the temperature and ionic strength dependence of this interaction in our model peptides was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in alpha- and beta-spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha-helix C region in spectrin are also involved in tetramer formation. Structural flexibility and lateral interactions may play a role in spectrin tetramerization.     

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.     

Timing and detuning studies of a hybridly mode-locked dye laser

The occurrence time of the output pulse of a hybridly mode-locked dye laser is studied. The good agreement between the experimental results and the theory serves to further verify the Maxwell-Schrodinger model used, and therefore serves to improve understanding of this ultrashort pulse laser. More importantly, from a practical standpoint, the studies directly address the issues of output pulse stability and timing jitter, two common problems in hybridly mode-locked dye lasers