Merocyanine 540 as a fluorescence indicator for molecular packing stress at the onset of lamellar-hexagonal transition of phosphatidylethanolamine bilayers

The fluorescence of Merocyanine 540 (MC 540) is sensitive to the molecular packing of membrane lipids. Therefore, the fluorescence of MC 540 is expected to be sensitive to the curvature-related packing stress at the onset of the lamellar-hexagonal phase transition. We measured the fluorescence intensity of MC 540 when the temperatures of lipid bilayers approached their lamellar-hexagonal phase transitions. The fluorescence of MC 540 in the presence of egg and dioleoylphosphatidylethanolamine bilayers increased at the respective lamellar-hexagonal phase transitions of these lipids. Furthermore, increases in fluorescence intensity were also observed at temperatures just below their phase transitions. The enhanced fluorescence was not due to the specific interaction of the dye with the ethanolamine headgroup, because no such increase was observed when the probe was exposed to phosphatidylethanolamines which do not form hexagonal phase within the range of applied temperature. In addition, when the temperature of the lamellar-hexagonal phase transition was shifted, by the addition of a small amount of phosphatidylcholine, the dependence of the fluorescence intensity on temperature was modified accordingly. We postulate that the change of MC 540 fluorescence intensity at temperatures approaching the lamellar-hexagonal phase transition reflects changes in the partition of MC 540 into the fluid lipid phase. The change in partition is influenced by the curvature stress in bilayers at temperatures just below the lamellar-hexagonal phase transition.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.     

Quantitative analytical electron microscopy of multiphase alloys

In this paper, we present a technique for analysis of composition gradients, using an analytical electron microscope, within the primary phase of a two-phase alloy for the case where the second-phase particle size is similar to the size of the irradiated volume. If the composition difference between the two phases is large, the detected compositional fluctuations associated with varying phase fractions may mask any underlying composition gradient of the primary phase. The analysis technique was used to determine grain boundary chromium concentration gradients in a nickel-base superalloy, alloy X-750. The technique may also be of use in other alloy systems.