A thermodynamic model for extended complexes of cholesterol and phospholipid

Studies of monolayer mixtures of certain phospholipids with cholesterol by epifluorescence microscopy and measurement of cholesterol desorption show evidence for the formation of "condensed complexes." A thermodynamic model of these complexes has been developed and has been shown to be generally consistent with observed phase diagrams, cholesterol desorption rates, and electric field susceptibility. Previous work has shown that complexes comprising 10-50 molecules provide good agreement with experimental results. The present study examines the calculated properties of complexes containing very large numbers of molecules and extends the condensed complex model to incorporate the formation of complexes of variable size. Trends in equilibrium composition are similar to those calculated for small complexes. Thermal transitions are continuous, with a strong composition dependence of the breadth of the transition. The average number of molecules in a large complex shows a pronounced dependence on the composition of the reaction mixture. Large complexes have properties of a separate thermodynamic phase.     

Two fatty acids can replace one phospholipid in condensed complexes with cholesterol

We report that the monolayer phase diagram for binary mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dihydrocholesterol (DChol) is largely unchanged when each phospholipid molecule is replaced by two myristic acid (MA) molecules or various mixtures of the lysophospholipid and myristic acid. The corresponding phase diagrams all show the formation of "condensed complexes" of DChol and lipid. The condensed complex stoichiometry is thus largely determined by the C14 fatty acid acyl chains, in this case about 4-4.6 per DChol molecule.     

Heavy metals in sewage sludges contribute to their adverse effects on the arbuscular mycorrhizal fungus<Emphasis Type="Italic">Glomus mosseae</Emphasis>

Applying sewage sludges to agricultural land is a widespread practice because of the sludges’ agronomic value as a source of plant nutrients and organic matter. Nevertheless, sludges often contain micropollutants that can constitute a menace for health and the environment. Arbuscular mycorrhizal fungi are sensitive to sewage sludges that have been spiked, or not, with metallic trace elements (MTE). Here we have investigated if MTE in sewage sludges could be responsible for effects on mycorrhizal development betweenGlomus mosseae andMedicago truncatula. The impact of a dehydrated or composted urban sewage sludge spiked or not with MTE, was tested on spore germination and root colonization byG. mosseae. The sewage sludges depressed both the presymbiotic andin planta stages of development of the mycorrhizal fungus. This negative effect was more related to the metallic pollutant contents of the sludges than to the presence of antagonistic microorganisms or phosphorus.     

Determination of rat oral bioavailability of soy-derived phytoestrogens using an automated on-column extraction procedure and electrospray tandem mass spectrometry

In recent years, consumption of herbal supplements as an alternative to pharmaceutical drug therapy has increased. For example, with the health claims labeling which describes the link between soy-protein and a reduced risk of coronary heart disease (CHD), the consumption of soy and soy-derived phytoestrogens has increased dramatically. That being said, the oral bioavailability of only a few soy phytoestrogens such as Daidzein and Genestein have been previously estimated. In this paper, we present the calculated percent of rat oral bioavailability of five soy-derived phytoestrogens (Genistein, Daidzein, Biochanin A, Coumestrol, and Zearalenone) in male Sprague-Dawley rats. The plasma quantitation required for the bioavailability calculation is performed by using a rapid on-line plasma extraction procedure for the quantitative analysis. To further speed up the analysis the rats were dosed using the 'n-in-one' (cassette) protocol. The rapid on-line extraction/quantitation methodology coupled to the cassette dosing analysis of phytoestrogens is the key point of this paper. The limit of quantitation (LOQ) for each compound was 1-1000 ng/ml with each plasma sample analysis taking less than 2 min. In general the percent oral bioavailability was determined to be between 11 and 28%.     

Optimization of a higher throughput microsomal stability screening assay for profiling drug discovery candidates

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.     

Histology of sterile male and female cones in<Emphasis Type="Italic"> Pinus monticola</Emphasis> (western white pine)

Two years of histological samples were collected from a Pinus monticola Dougl. (western white pine) tree identified as not producing mature pollen or seed cones. Anatomical information was collected to the ultrastructural level, to assess possible mechanisms for pollen and cone abortion resulting in sterility. Development of male and female gametophytes in the sterile western white pine tree was arrested after meiosis and before further cell divisions could take place. Sterile male gametophytes (pollen grains) had poorly developed pollen walls and sacci, reduced and degenerative cytoplasm, and no evidence of stored starch grains. The pollen cone aborted prior to pollen dehiscence. Meiosis of the megaspore mother cell in the ovule produced four megaspores, but development was stopped at the functional megaspore stage. The seed cone aborted in the first year of growth before winter dormancy. Tapetal tissue in sterile microsporangia appeared similar to that of fertile microsporangia, until the vacuolate, uninucleate microspore stage. Tapetal cells and thecal fluid surrounding the sterile microspores persisted well past the time when microsporangia on fertile trees started the process of maturation and desiccation. At pollen dehiscence, sterile pollen cones did not release any pollen and the microsporangia were filled with a sticky fluid. The behaviour of the tapetum in P. monticola sterile cones is compared with reports of tapetal function and malfunction reported in studies of angiosperm and other gymnosperm species. The occurrence and timing of gametophyte abortion in both cone sexes suggests a genetic rather than environmental basis for the sterility mechanism.     

FGF signaling through FGFR1 is required for olfactory bulb morphogenesis

During development, the embryonic telencephalon is patterned into different areas that give rise to distinct adult brain structures. Several secreted signaling molecules are expressed at putative signaling centers in the early telencephalon. In particular, Fgf8 is expressed at the anterior end of the telencephalon and is hypothesized to pattern it along the anteroposterior (AP) axis. Using a CRE/loxP genetic approach to disrupt genes in the telencephalon, we address the role of FGF signaling directly in vivo by abolishing expression of the FGF receptor Fgfr1. In the Fgfr1-deficient telencephalon, AP patterning is largely normal. However, morphological defects are observed at the anterior end of the telencephalon. Most notably, the olfactory bulbs do not form normally. Examination of the proliferation state of anterior telencephalic cells supports a model for olfactory bulb formation in which an FGF-dependent decrease in proliferation is required for initial bulb evagination. Together the results demonstrate an essential role for Fgfr1 in patterning and morphogenesis of the telencephalon.     

Progressive restriction in fate potential by neural progenitors during cerebral cortical development

During early stages of cerebral cortical development, progenitor cells in the ventricular zone are multipotent, producing neurons of many layers over successive cell divisions. The laminar fate of their progeny depends on environmental cues to which the cells respond prior to mitosis. By the end of neurogenesis, however, progenitors are lineally committed to producing upper-layer neurons. Here we assess the laminar fate potential of progenitors at a middle stage of cortical development. The progenitors of layer 4 neurons were first transplanted into older brains in which layer 2/3 was being generated. The transplanted neurons adopted a laminar fate appropriate for the new environment (layer 2/3), revealing that layer 4 progenitors are multipotent. Mid-stage progenitors were then transplanted into a younger environment, in which layer 6 neurons were being generated. The transplanted neurons bypassed layer 6, revealing that layer 4 progenitors have a restricted fate potential and are incompetent to respond to environmental cues that trigger layer 6 production. Instead, the transplanted cells migrated to layer 4, the position typical of their origin, and also to layer 5, a position appropriate for neither the host nor the donor environment. Because layer 5 neurogenesis is complete by the stage that progenitors were removed for transplantation, restrictions in laminar fate potential must lag behind the final production of a cortical layer. These results suggest that a combination of intrinsic and environmental cues controls the competence of cortical progenitor cells to produce neurons of different layers.     

pH stability of HLA-DR4 complexes with antigenic peptides

Complexes between antigenic peptides and class II proteins of the major histocompatibility complex (MHC) trigger cellular immune responses. These complexes usually dissociate more rapidly at mildly acidic pH, where they are formed intracellularly, as compared to neutral pH, where they function at the cell surface. This paper describes the pH dependence of the dissociation kinetics of complexes between MHC proteins and antigenic peptides containing aspartic and glutamic acid residues. Some of these complexes show an unusual pH dependence, dissociating much more rapidly at pH 7 than at pH 5.3. This occurs when the carboxylate group of the aspartic or glutamic acid residue is located in a neutral pocket of the protein. In contrast, solvent-exposed carboxylate groups or carboxylate groups buried in pockets where they form salt bridges with the protein do not show this unusual pH dependence. The kinetic data having the unusual pH dependence conform closely to a model in which there is a rapid reversible equilibration between a less stable deprotonated complex and a more stable protonated complex. In this model, the pK(a) of the protonation reaction for the partially buried peptide carboxylate group ranges from 7.7 to 8.3, reflecting the strongly basic conditions required for deprotonation. One of the few peptide/MHC complexes demonstrated to play a role in autoimmunity in humans contains a buried peptide carboxylate and shows this unusual pH dependence. The relevance of this finding to understanding the chemical basis of autoimmunity is briefly discussed.     

DNA structure: what's in charge?

DNA structure is well known to be sensitive to hydration and ionic strength. Recent theoretical predictions and experimental observations have raised the idea of the intrusion of monovalent cations into the minor groove spine of hydration in B-form DNA. To investigate this further, extensions and further analysis of molecular dynamics (MD) simulations on d(CGCCGAATTCGCG), d(ATAGGCAAAAAATAGGCAAAAATGG) and d(G(5)-(GA(4)T(4)C)(2)-C(5)), including counterions and water, have been performed. To examine the effective of minor groove ions on structure, we analyzed the MD snapshots from a 15 ns trajectory on d(CGCGAATTCGCG) as two subsets: those exhibiting a minor groove water spine and those with groove-bound ions. The results indicate that Na(+) at the ApT step of the minor groove of d(CGCCGAATTCGCG) makes only small local changes in the DNA structure, and these changes are well within the thermal fluctuations calculated from the MD. To examine the effect of ions on the differential stability of a B-form helix, further analysis was performed on two longer oligonucleotides, which exhibit A-tract-induced axis bending localized around the CpG step in the major groove. Plots of axis bending and proximity of ions to the bending locus were generated as a function of time and revealed a strong linear correlation, supporting the idea that mobile cations play a key role in local helix deformations of DNA and indicating ion proximity just precedes the bending event. To address the issue of "what's in charge?" of DNA structure more generally, the relative free energy of A and B-form d(CGCGAATTCGCG) structures from MD simulations under various environmental circumstances were estimated using the free energy component method. The results indicate that the dominant effects on conformational stability come from the electrostatic free energy, but not exclusively from groove bound ions per se, but from a balance of competing factors in the electrostatic free energy, including phosphate repulsions internal to the DNA, the electrostatic component of hydration (i.e. solvent polarization), and electrostatic effects of the counterion atmosphere. In summary, free energy calculations indicate that the electrostatic component is dominant, MD shows temporal proximity of mobile counterions to be correlated with A-track-induced bending, and thus the mobile ion component of electrostatics is a significant contributor. However, the MD structure of the dodecamer d(CGCGAATTCGCG) is not highly sensitive to whether there is a sodium ion in the minor groove.