Design and implementation of an ribonucleic acid (RNA) directed fragment library.

The design of RNA binding ligands is complicated by issues of specificity, target flexibility, and the tractability of known RNA inhibitors toward chemical derivitization. To address these difficulties, an RNA-directed fragment compound library is presented. We began with an analysis of 120 small molecules with reported RNA-binding activity.

Monitoring the effect of three humic acids on a model membrane system using 31P NMR.

The sorption of three humic acids to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine multilamellar vesicle model membrane systems was studied by phosphorus nuclear magnetic resonance (31P NMR). The effects of temperature and pH were investigated. The gel --> bilayer transition did not appear to be affected by any of the humic acids at pH 7; however, all three humic acids induced a perturbation to this transition and to the bilayer structure at pH 4.

V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients.

The metabolic downregulation critical for long-term survival of Artemia franciscana embryos under anoxia is mediated, in part, by a progressive intracellular acidification. However, very little is known about the mechanisms responsible for the pH transitions associated with exposure to, and recovery from, oxygen deprivation. In the present study, we demonstrate with 31P-NMR that incubation of intact embryos with the V-ATPase inhibitor bafilomycin A1 severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphate levels.

Chromophore formation in resorcinarene solutions and the visual detection of mono- and oligosaccharides.

The colorimetric properties of resorcinarene solutions had not been investigated since Baeyer's initial synthesis. We recently reported that solutions containing resorcinarene macrocycles develop color upon heating or standing. In the presence of saccharides, these solutions exhibit significant color changes which are easily seen.

Infrared spectroscopy of human apolipoprotein fragments in SDS/D2O: relative lipid-binding affinities and a novel amide I assignment.

Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D2O. Five of the peptides correspond to proposed lipid-binding domains of human apolipoproteins [apoC-I(7-24), apoC-I(35-53), apoA-II(18-30)+, apoA-I(166-185), apoE(267-289)], and the sixth is the de novo lipid associating peptide LAP-20. The amide I infrared absorption patterns are generally consistent with predominantly helical structures (as determined previously by NMR spectroscopy and distance geometry calculations) and further suggest that apoA-I(166-185) and apoE(267-289) are bound to SDS relatively weakly in comparison to the other four peptides.

Conformations of human apolipoprotein E(263-286) and E(267-289) in aqueous solutions of sodium dodecyl sulfate by CD and 1H NMR.

Structures of apoE(263-286) and apoE(267-289) have been determined in aqueous solution containing 90-fold molar excess of perdeuterated sodium dodecyl sulfate by CD and 1H NMR. Conformations were calculated by distance geometry based on 370 and 276 NOE distance restraints, respectively. RMSD for superimposing the region 265-284 from an ensemble of 41 structures for apoE(263-286) is 0.

Conformation of human serum apolipoprotein A-I(166-185) in the presence of sodium dodecyl sulfate or dodecylphosphocholine by 1H-NMR and CD. Evidence for specific peptide-SDS interactions.

The segment, YSDELRQRLAARLEALKENG, corresponding to residues 166 to 185 of human serum apolipoprotein A-I, was studied by circular dichroism and NMR spectroscopy in sodium dodecyl sulfate and dodecylphosphocholine micelles. 2-Dimensional NOESY, TOCSY and DQF-COSY spectra of apoA-I(166-185) in perdeuterated sodium dodecyl sulfate (SDS-d25) and dodecylphosphocholine (DPC-d38) micelles were collected at a peptide/SDS (DPC) ratio of 1:40. Similar CD spectra and NOE connectivity patterns were observed for apoA-I(166-185) in SDS and DPC, indicating a similar helical conformation in both.

Structural studies of a peptide activator of human lecithin-cholesterol acyltransferase.

The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase (LCAT) despite its lack of sequence homology to apolipoprotein A-I, the primary in vivo activator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP-20's ability to activate LCAT were studied by optical and two-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles.

An alternate apoprotein conformation in high density apolipoprotein discoidal complexes. A Fourier transform infra-red study.

Discoidal complexes have been prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the apoproteins of HDL3 (apo HDL3) or purified apo A-I. Gel electrophoresis established that apo HDL3 contained 74% apo A-I. Deconvolution and curve-fitting of the infra-red amide I band of the apoprotein in the lipid-protein complex revealed a secondary structure containing approximately 40% alpha-helix and 50% beta-structure.

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study.

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.

Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins.

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was approximately 0.

Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins.

Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6-20 mol%) and into VLDL (7-10 mol%).

Lipid-lipid interactions in reconstituted high-density lipoproteins by deuterium nuclear magnetic resonance.

Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted high-density lipoproteins (rHDLs) composed of apoHDL3 with either dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg PC) as the monolayer and either cholesteryl oleate (CO) or triolein (TO) as the core. The effect of the monolayer on the core was observed by deuterium nuclear magnetic resonance (2H NMR) studies of rHDLs containing the core component cholesteryl [18, 18, 18-2H3] oleate [( 2H3]CO) or tri[16,16-2H2]oleoylglycerol [( 2H6]TO) surrounded by a monolayer of either DPPC or egg PC as a function of temperature. The reverse effect, that of the core on the monolayer, was examined by both 2H and 31P NMR studies of rHDLs containing [5,5-2H2]PC in the presence of CO or TO as a function of temperature.

Surface diffusion in human serum lipoproteins.

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.

Orientational order of cholesteryl oleate in low-density lipoprotein observed by 2H-NMR.

Cholesteryl oleate, selectively deuterated at various positions along the acyl chain, has been incorporated into fresh human serum low-density lipoprotein (LDL2). Temperature-dependent 2H-NMR spectra were recorded between 15 and 45 degrees C. For deuterons at C-2' and C-5' of the acyl chain, two 2H-NMR spectral components, a broad and a narrow signal, are observed.

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes.

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidylcholine (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates, 1/T1,SL, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.