Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation

Melanocytes in the skin are derived from the embryonic neural crest. Recently, mutations in endothelin 3 and the endothelin receptor B genes have been shown to result in gross pigment defects, indicating that this signalling pathway is required for melanocyte development. We have examined the effects of endothelins on melanocyte progenitors in cultures of mouse neural crest. Firstly, they stimulate an increase in progenitor number and act synergistically with another factor, Steel factor, in the survival and proliferation of the progenitors. These findings are consistent with findings from mice with natural mutations in the endothelin receptor B gene, which show an early loss of melanocyte progenitors. Secondly, endothelins induce differentiation of the progenitors into fully mature pigmented melanocytes. This finding is consistent with the expression of endothelins in the skin of mice at the initiation of pigmentation. The melanocytes generated in endothelin-treated cultures also become responsive to alpha melanocyte-stimulating hormone, which then acts to regulate the activity of the pigmentation pathway. These findings indicate two key roles for endothelin in melanocyte development: regulation of expansion of the progenitor pool and differentiation of progenitors into mature melanocytes.     

Effect of a single high-fat meal on endothelial function in healthy subjects

Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL-60 cells were analyzed using 3H-labeled GM3 ([3H]GM3). [3H]GM3 was rapidly internalized into the cells (trypsin-resistant fraction) 8 times more than the control, 3H-labeled GM1 ([3H]GM1). In addition, not only incorporation but also metabolism of [3H]GM3 was more rapid than [3H]GM1 in HL-60 cells. Moreover, one of the metabolites was found to co-migrate with ceramide in thin-layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation-inducible GM3 in HL-60 cells rather than from non-inducing GM1.     

Interaction of biotin with streptavidin. Thermostability and conformational changes upon binding

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.     

Brownian motion on a square Lennard-Jones lattice: Trapping, hopping, and diffusion

Previously we showed that cGMP hydrolysis in rat whole retinal homogenates exhibited a dose-dependent inhibition following developmental lead exposure and a concentration-dependent inhibition with direct Pb2+ exposure. Additionally, developmental lead exposure resulted in a dose-dependent increase in retinal cGMP and rod Ca2+ levels. To determine whether Pb2+ or Ca2+ directly inhibited the rod-specific cGMP phosphodiesterase (PDE) and to examine the kinetic mechanism of this inhibition, purified bovine rod cGMP PDE was assayed in the presence of varying concentrations of cGMP, and Mg2+, Pb2+, and/or Ca2+. Increasing concentrations of the substrate, cGMP, resulted in a shift of the Pb2+ and Ca2+ concentration-response curves to the left, indicating a decrease in the half-maximal inhibitory concentrations of Pb2+ from nanomolar to picomolar levels. Increasing concentrations of the cofactor, Mg2+, resulted in a shift of the Pb2+ and Ca2+ concentration-response curves to the right, indicating a decrease in the inhibition of PDE activity by Pb2+ or Ca2+. A plot of 1/velocity vs 1/Mg2+ as a function of Pb2+ revealed that picomolar concentrations of Pb2+ competitively inhibited PDE relative to millimolar concentrations of Mg2+. Consistent with this finding, Mg2+ reversed the Pb(2+)-induced inhibition of PDE. Our recent kinetic analysis showed that Mg2+ and cGMP bind at interacting sites on the PDE in a random order. The present results reveal that Pb2+ may bind at the same site but with 4-6 log units higher affinity than Mg2+, thus preventing the hydrolysis of cGMP. These findings provide a novel mechanism for understanding the Pb(2+)-induced inhibition of cGMP PDE. These results may have implications for other enzymes using Mg2+ as a cofactor and suggest that Mg2+ may be useful in these situations for reversing the inhibition by Pb2+.     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.