Production of <Emphasis Type="Italic">in vitro</Emphasis> amplified DNA pseudolibraries and high-throughput cDNA target amplification

Background  

Many structural biology- and high-throughput laboratories experience the acquisition of multiple cDNAs from different sources as a rather time- and resource-consuming procedure. The techniques presented here solve these problems.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

Semiautomated processing of systolic time intervals.

Systolic time intervals are commonly used to identify changes in ventricular function. The method described facilitates measurement and calculation of many such intervals. This method utilizes a printed polygraph recording of electrocardiogram, phonocardiogram, and carotid pulse contour; a digitizing device for reading the necessary coordinates from the record; and a minicomputer for calculation of the intervals and for data analysis. Intervals related to approximately 100 pulse beats can be read, calculated, printed in chart and graph form, and subjected to some analyses in about an hour.     

Structural characterization of nanoscale meshworks within a nucleoporin FG hydrogel

The permeability barrier of nuclear pore complexes (NPCs) controls all exchange of macromolecules between the cytoplasm and the cell nucleus. It consists of phenylalanine-glycine (FG) repeat domains apparently organized as an FG hydrogel. It has previously been demonstrated that an FG hydrogel derived from the yeast nucleoporin Nsp1p reproduces the selectivity of authentic NPCs. Here we combined time-resolved optical spectroscopy and X-ray scattering techniques to characterize such a gel. The data suggest a hierarchy of structures that form during gelation at the expense of unstructured elements. On the largest scale, protein-rich domains with a correlation length of ~16.5 nm are evident. On a smaller length scale, aqueous channels with an average diameter of ~3 nm have been found, which possibly represent the physical structures accounting for the passive sieving effect of nuclear pores. The protein-rich domains contain characteristic β-structures with typical inter-β-strand and inter-β-sheet distances of 1.3 and 0.47 nm, respectively. During gelation, the formation of oligomeric associates is accompanied by the transfer of phenylalanines into a hydrophobic microenvironment, supporting the view that this process is driven by a hydrophobic collapse.     

Aging sensitizes toward ROS formation and lipid peroxidation in PS1M146L transgenic mice

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19-22 months) PS1M146L transgenic animals compared to PS1wt mice and not in young (3-4 months) or middle-aged mice (13-15 months). Similarly, in splenic lymphocytes expressing the transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes, indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.     

Prolonged submaximal exercise and L-carnitine in humans

Changes in the main physiological parameters and circulating indicators of carbohydrate, protein, lipid (and ketone body) metabolism were measured in ten exercising subjects before L-carnitine (L-carn) loading, after 4 weeks of daily loading with 2 g L-carn, and 6-8 weeks after terminating L-carn administration. Measurements were made on venous blood samples collected during each experiment at fixed time intervals over an initial rest of 45 min, 60 min bicycle exercise performed near 50% VO2max and 120 min recovery. Free and total plasma carnitine levels reached a plateau corresponding to an average rise of 25% for both fractions, 9-10 days after the beginning of the L-carn diet. These levels returned to their initial values 6-8 weeks after cessation of the supply. Generally L-carn supplementation did not significantly modify the physiological parameters and circulating metabolites. No distinct increase of the relative participation of endogenous lipids in the fuel supply of prolonged submaximal exercise was observed. In normal human subjects the increased demand for fatty acid oxidation resulting from exercise seems to be adequately supported by endogenous levels of carnitine.     

Soil respiration does not acclimatize to warmer temperatures when modeled over seasonal timescales

Soil warming studies have generally demonstrated an ephemeral response of soil respiration to warming suggesting acclimatization to increased temperatures. Many of these studies depict acclimatization as an empirical temperature-respiration model with data collected from late spring through early autumn. We examined the apparent temperature sensitivity of soil respiration in chronically warmed soils over three different timescales: annually, during the growing season, and seasonally during winter, spring, summer, and fall. Temperature sensitivity was evaluated by fitting exponential and flexible temperature functions as mixed effects models. From model coefficients, we estimated annual, growing season, and season-specific Q ₁₀ values, and assessed the ability of model coefficients to predict daily soil respiration rates over a two-year period. We found that respiration in warmed soils can exhibit characteristics of acclimatized temperature sensitivity depending on the timeframe and the function (exponential or flexible) used. Models using growing season data suggested acclimatization while models using data collected in winter or spring indicated enhanced temperature sensitivity with 5 °C of warming. Differences in temperature sensitivity affected predicted daily soil respiration rates, particularly in winter and spring. Models constructed over longer timescales overestimated daily respiration rates by as much 10–40 % whereas season-specific predictions were generally within 2 % of actual values. Failure to use season-specific models to depict changes in temperature dependence may over- or under-estimate carbon losses due to climate warming, especially during the colder months of the year.     

Free D- and L-amino acids in ventricular cerebrospinal fluid from Alzheimer and normal subjects

Summary Free D-Ser, D-Asp and total D-amino acids were significantly higher (p < 0.05) in Alzheimer (AD) ventricular CSF than in normal CSF. There was no significant difference in the total L-amino acids between AD and normal CSF, but L-Gln and L-His were significantly higher (p < 0.05) in ADCSF. The higher concentrations of these D- and L-amino acids in AD ventricular CSF could reflect the degenerative process that occurs in Alzheimer's brain since ventricular CSF is the repository of amino acids from the brain.     

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary,multi‐scale approach

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear‐cutting. Retention of habitat trees and deadwood has since become common practice also in continuous‐cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade‐offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi‐functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio‐economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.