Intermolecular protein interactions in solutions of bovine lens beta L-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

We report the magnetic field dependence of 1/T1 of solvent water protons and deuterons (nuclear magnetic relaxation dispersion, or NMRD, profiles) for solutions of steer lens beta L-crystallin. Such data allow the study of intermolecular protein interactions over a wide concentration range, here 1-34% vol/vol, by providing a measure of the rotational relaxation time of solute macromolecules. We conclude that, for approximately less than 5% protein, the solute particles are noncompact, with a rotationally averaged volume approximately three times that of a compact 60-kD sphere. (Earlier results for alpha-crystallin, approximately 1,000 kD, from optical and osmotic measurements (Vérétout and Tardieu, 1989. J. Mol. Biol. 205:713-728), show a similar, approximately twofold, effect). At intermediate concentrations, to approximately 20% protein, there is evidence for limited association or oligomerization, as found for the structurally related gamma II-crystallin (Koenig et al. 1990. Biophys. J. 57:461-469), to a limiting size about two-thirds that of alpha-crystallin. The difference in NMRD behavior of the three classes of crystallins is consonant with their differing osmotic properties (Vérétout and Tardieu. J. Mol. Biol. 1989, 205:713-728; Kenworthy, McIntosh, and Magid. Biophys. J. 1992. 61:A477; Tardieu et al. 1992. Eur. Biophys. J. 21:1-12). We indicate how the unusual structures and interactions of these three classes of proteins can be combined to optimize transparency and minimize colloid osmotic difficulties in eye lens.     

Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol).

The interactive properties of liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are of interest because such liposomes are being developed as drug delivery vehicles and also are ideal model systems for measuring the properties of surface-grafted polymers. For bilayers containing PEG-lipids with PEG molecular weights of 350, 750, 2000, and 5000, pressure-distance relations have been measured by X-ray diffraction analysis of liposomes subjected to known applied osmotic pressures. The distance between apposing bilayers decreased monotonically with increasing applied pressure for each concentration of a given PEG-lipid. Although for bilayers containing PEG-350 and PEG-750 the contribution of electrostatic repulsion to interbilayer interactions was significant, for bilayers containing PEG-2000 and PEG-5000 the major repulsive pressure between bilayers was a steric pressure due to the attached PEG. The range and magnitude of this steric pressure increased both with increasing PEG-lipid concentration and PEG size, and the extension length of the PEG from the bilayer surface at maximum PEG-lipid concentration depended strongly on the size of the PEG, being less than 35 A for PEG-750, and about 65 A for PEG-2000 and 115 A for PEG-5000. The measured pressure-distance relations have been modeled in terms of current theories (deGennes, 1987; Milner et al., 1988b) for the steric pressure produced by surface-grafted polymers, as modified by us to take into account the effects of polymer polydispersity and the possibility that, at low grafting densities, polymers from apposing bilayers surfaces can interpenetrate or interdigitate. No one theoretical scheme is sufficient to account for all the experimental results. However, for a given pressure regime, PEG-lipid size, and PEG-lipid surface density, the appropriately modified theoretical treatment gives a reasonable fit to the pressure-distance data.     

Evidence for the influence of seagrasses on the benthic nitrogen cycle in a coastal plain estuary near Beaufort,North Carolina (USA)

A study was undertaken to evaluate the interrelationship between the presence of seagrasses, Zostera marina and Halodule wrightii, and the physical and chemical properties of sediments in a coastal plain estuary near Beaufort, North Carolina. In sediments underlying a cover of seagrass, silt-clay, organic matter, exchangeable ammonium, ammonium dissolved in pore waters and total nitrogen were larger than in unvegetated profiles. The magnitude of the physical and chemical properties of sediments varied according to the location of the station in relation to the vegetation, as well as the continuity in the distribution of the seagrass. The largest pools of nitrogen, the finest sediment texture, and the greatest organic matter content were in sediments associated with the mid bed regions of seagrass meadows, intermediate at the edges of the bed and small isolated patches of grass, and least in unvegetated substrate.General conclusions from this study are: 1) once established, seagrasses appear capable of modifying the sediment texture as well as the organic matter and nitrogen content; 2) nitrogen accumulates beneath the vegetation suggesting that vegetated sediments are sinks; however, functional recycling mechanisms seem to be operating as suggested by the larger magnitude of remineralized nitrogen in the vegetated profiles; and 3) the establishment of seagrasses in this geographical region are not necessarily restricted by the sediment properties measured in this study. These data and conclusions are discussed in regard to an application of contemporary theories of ecosystem development to seagrass systems.Contribution Number 82-22-B     

Peering inside lipid rafts and caveolae

Clustering of proteins into membrane microdomains, such as lipid rafts and caveolae, could act as a mechanism for regulating cell signaling and other cellular functions. Certain lipid modifications are hypothesized to target proteins to these domains on the cytoplasmic leaflet of the plasma membrane. This concept has now been tested in living cells using an assay sensitive to the lateral distribution of proteins in membranes over sub-micron distances.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

不同耕作措施对冬小麦-夏玉米复种连作系统土壤有机碳和水分利用效率的影响

在连续8年田间定位试验的基础上,分析了关中平原冬小麦 夏玉米复种连作系统2008—2009年连续两个生长季期间不同耕作措施(结合秸秆还田和不还田)对土壤有机碳和水分利用率的影响.结果表明: 相对于传统耕作,保护性耕作有利于土壤有机碳、水分利用效率和作物产量的提高,其中在“深松+秸秆还田”耕作模式下的增幅最高,土壤有机碳含量在0～30 cm土层增幅达到19.5%,水分利用效率和作物产量提高了16.9%和20.5%,而免耕模式则有效提高了0～10 cm土层有机碳含量.在该地区土壤和气候条件下,深松结合秸秆粉碎还田是最理想的耕作模式,最有利于土壤有机碳累积,并提高水分利用效率和作物产量.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

Validation of Normalizations,Scaling, and Photofading Corrections for FRAP Data Analysis

Fluorescence Recovery After Photobleaching (FRAP) has been a versatile tool to study transport and reaction kinetics in live cells. Since the fluorescence data generated by fluorescence microscopy are in a relative scale, a wide variety of scalings and normalizations are used in quantitative FRAP analysis. Scaling and normalization are often required to account for inherent properties of diffusing biomolecules of interest or photochemical properties of the fluorescent tag such as mobile fraction or photofading during image acquisition. In some cases, scaling and normalization are also used for computational simplicity. However, to our best knowledge, the validity of those various forms of scaling and normalization has not been studied in a rigorous manner. In this study, we investigate the validity of various scalings and normalizations that have appeared in the literature to calculate mobile fractions and correct for photofading and assess their consistency with FRAP equations. As a test case, we consider linear or affine scaling of normal or anomalous diffusion FRAP equations in combination with scaling for immobile fractions. We also consider exponential scaling of either FRAP equations or FRAP data to correct for photofading. Using a combination of theoretical and experimental approaches, we show that compatible scaling schemes should be applied in the correct sequential order; otherwise, erroneous results may be obtained. We propose a hierarchical workflow to carry out FRAP data analysis and discuss the broader implications of our findings for FRAP data analysis using a variety of kinetic models.