An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end ofa hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 370C. For reaction (19, 20). Synthetic details will be provided elsewhere.Measurement of Thermodynamic Parameters. The buffer for melting experiments was 1 M NaCl/10 mM sodium cacodylate/0.5 mM EDTA. Absorbance versus temperature curves were measured at 280 nm with a heating rate of 10C/min. These curves were analyzed by fitting to a two-state model with sloping base lines through use of a nonlinear least-squares program (21).Predictions of Secondary Structure. Sets of 20 secondary structures were generated with the program of Zuker (12), kcal/mol, and internal loops of three were given a AG'7 for loop closure of 5.1 + 0.3 = 5.4 kcal/mol (23). (iii) Stacking and pairing of terminal mismatches were included in calculating free energies of internal loops in the following manner. Single mismatches were given AG7 = 0.8 kcal/mol. For larger internal loops, terminal GA, UU, and other mismatches adjacent to COG pairs were given favorable free energy increments of -2.7, -2.5, and -1.5 kcal/mol, respectively (24). These mismatches adjacent to A-U pairs were given AG' values of -2.
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Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.
Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-sidedown" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2 H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.Cholesterol constitutes a major component of mammalian cellular membranes. Its correct distribution among intracellular membranes and the plasma membrane is essential for the homeostasis of mammalian cells. Thus, intracellular trafficking plays a major role in the correct disposition of internalized cholesterol and in the regulation of cholesterol efflux (for a recent review, see Ref.
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