Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

Solution conformations of cyclosporins and magnesium-cyclosporin complexes determined by vibrational circular dichroism

Vibrational circular dichroism (VCD) spectroscopy was used to investigate the solution conformations of cyclosporins A, C, D, G, and H in CDCl(3), in the amide I and NH/OH-stretching regions, and their corresponding magnesium complexes in CD(3)CN, in the amide I region. VCD spectra are sensitive to the chiral arrangement of Cdbond;O and NH bonds in this cyclic undecapeptide. Calculations of molecular geometries, as well as IR and VCD intensities of model cyclosporin fragments that include the intramolecular hydrogen bonds of the crystal conformations of cyclosporins A and H (CsA and CsH), were carried out at the density functional theory (DFT; BPW91 functional/6-31G* basis set) level. The good agreement between IR and VCD spectra from experiment and DFT calculations provides evidence that the crystal conformation of CsA is dominant in CDCl(3) solution; CsH, however, assumes both an intramolecularly hydrogen-bonded crystal conformation and more open forms in solution. Comparisons of the experimental and calculated VCD spectra in the NH/OH-stretching region of the noncomplexed cyclosporins indicate that conformers with both free and hydrogen-bonded NH and OH groups are present in solution. Differences between the IR and VCD spectra for the metal-free and magnesium-complexed cyclosporins are indicative of strong interactions between cyclosporins and magnesium ions.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

On the uptake of nystatin by saccharomyces cerevisiae. 5 The release of amino acids

The release of amino acids from Saccharomyces cerevisiae under the action of nystatin has been studied. The kinetic results do not show any dependence upon molecular size but do support earlier results suggesting that interactions depend upon slow diffusion processes through the cell wall. The effects of temperature, sterol concentration and the presence of calcium ion have also been investigated.     

Phase transition properties of 1,2- and 1,3-diacylphosphatidylethanolamines with modified head groups

The phase transition properties of dilute aqueous suspensions of "nonhydrated" (i.e., lipid suspensions which had not been heated above room temperature or above the main phase transition temperature of the fully hydrated lipid, whichever was lower) and hydrated 1,2(alpha)- and 1,3(beta)-dipalmitoylphosphatidylethanolamines with modified head groups have been determined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. In both the 1,2 and 1,3 series, the head-group modifications of the phosphoethanolamine moiety included N-methyl, N,N-dimethyl, and N,N,N-trimethyl (phosphocholine). In the 1,2 series, additional modifications were dinitrophenyl, trinitrophenyl, N-(dinitrophenyl)aminocaproyl, N-(trinitrophenyl)aminocaproyl, and N-4-nitro-2,1,3-benzoxadiazole. Also included in this study were 1,2-dihexadecylphosphatidylethanolamine and the corresponding N-methyl-substituted lipid. In general, increasing bulkiness of the head-group substituent caused increasing lowering of the transition temperature, the most extreme cases among the hydrated lipids being the 45 degrees C lowering produced by the N-(dinitrophenyl)aminocaproyl substitution and its trinitrophenyl analogue in the 1,2 series. No simple trend is evident in the changes produced in the calorimetric enthalpy of transitions.     

Thermodynamics of phospholipid-sucrose interactions.

The effect of 0-1.0 M sucrose on the phase-transition properties of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (1,2-DPPC) was examined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. Increasing the concentration of sucrose caused a small, but experimentally significant, increase in the temperature (Tm) of maximal excess apparent specific heat (Cmax) and in delta T 1/2 (the transition width at 1/2 Cmax), a reduction in Cmax, and a small decrease (approximately 8-10% at 1.0 M sucrose compared with 0 M sucrose) in the calorimetric enthalpy (delta Hcal) of the gel-to-liquid crystalline transition. The calorimetric parameters of the pretransition of 1,2-DPPC were not significantly affected by sucrose in the concentration range examined, except there was a 1.0 degree C increase in the temperature (Tp) of maximal excess apparent specific heat in the presence of 1.0 M sucrose. The results are discussed in terms of the possible molecular mechanisms that could have caused the observed changes and are contrasted with the results obtained by C. -H. Chen et al. (1981, Biophys. J., 36:359-367).     

Storage protein profiles in Spanish and runner market type peanuts and potential markers

Background  

Proteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE), coupled with highly sensitive mass spectrometry (MS), and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies.     

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]₂; Poly dG – Poly dC; [Poly(dGdC)]₂), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values. Received: 16 April 1997 / Accepted: 9 July 1997