A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA from Animal Tissues and Cell Cultures

A two step procedure for the quantitative isolation of protein- and RNA-free double-strand DNA from animal tissue and cell homogenates is described. In the first step proteins not complexed with DNA are hydrolyzed with an immobilized protease (Proteinase K) that is separated by filtration after the de-proteinization. Then the DNA is adsorbed to hydroxylapatite (HA) and desorbed from the adsorbent by stepwise elution with buffers of increasing ionic strength. The DNA content was determined directly from the absorption at 260 nm. The melting curve of the isolated DNA showed that it was double stranded. The protein content in the DNA was determined from the ratio of the adsorbance at 260 to 230 nm. Non-histone proteins complexed to DNA determined the rate of deproteinization that was found to be tissue specific. These proteins were found to have a larger influence on the ratio A₂₆₀/A₂₃₀ than histones, indicating that their absorption (at 230 nm) is markedly perturbed when they are bound to DNA.     

Osmoregulation in a nectar-feeding insect, the carpenter bee Xylocopa capitata: water excess and ion conservation

Abstract The liquid diet and high metabolic water production during flight in the carpenter bee Xylocopa capitata Smith 1854 (Hymenoptera, Anthophoridae) causes a water excess, and this is exacerbated by a low dietary intake of ions. The nectar and pollen of the preferred food-plants, Virgilia divaricata Adamson and Podalyria calyptrata Willd., and other Fabaceae had low levels of sodium. Analyses of the bees and their body fluids showed that the bees have an exceptionally low Na content, and Na homeostasis seems to depend on recycling almost all Na which enters the rectum. The copious dilute urine (137 mOsm) had Na and K concentrations of only 3.4 and 7.0 ITIM, respectively. Isolated preparations of Xylocopa Malpighian tubules secreted a fluid with a K concentration 10 times that of the haemolymph. This means that recycling of K is as important as that of Na, and the bulk of K resorption probably occurs passively in the ileum. This study is the first to examine hymenopteran Malpighian tubules. Their stimulation by cAMP is indicative of the presence of a diuretic hormone in Xylocopa.     

Rigidity loss of protein macromolecule induced by force—Effective field theory

In the framework of the effective field theory for the order parameter, which characterizes the degree of deviating the protein globule structure from its native state, the phase transition of the protein macromolecule from the elastic state into the plastic one under its mechanical stretching is considered. Elastic properties of a protein are studied as a function of the applied force, temperature, and the mean coordination number of the protein “network.” Proteins 2014; 82:966–974. © 2013 Wiley Periodicals, Inc.     

Hydrolysis of insoluble cellulose to glucose catalyzed by cellulase‐containing liposomes in an aqueous solution of 1‐butyl‐3‐methylimidazolium chloride

The liposome containing cellulase from Trichoderma viride was prepared under the condition that an appreciable amount of cellulase was incorporated in lipid membranes. The liposomal cellulase and free enzyme were examined in their hydrolytic activities to insoluble cellulose powder CC31 in the acetate buffer solution (pH 4.8) of 15 w/w% [Bmim][Cl] (1‐butyl‐3‐methylimidazolium chloride). The mean diameter and size distribution of cellulase‐containing liposome were practically unchanged under the above condition. The free cellulase was deactivated more rapidly than the liposomal cellulase in catalyzing the hydrolysis of 2.0 g/l CC31 at 45°C in the presence of [Bmim][Cl] for 48 h. The activities of liposomal and free cellulase to cellobiose as soluble substrate were less susceptible to [Bmim][Cl] than their cellulolytic activities to CC31, meaning that β‐glucosidase is relatively stable among the three enzyme components of cellulase. The rate of glucose production could be appreciably improved by the pretreatment of CC31 with [Bmim][Cl] alone at 120°C for 30 min followed by the liposomal cellulase‐catalyzed hydrolysis of the substrate at 45°C at the [Bmim][Cl] concentration of 15 w/w%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1190–1196, 2013     

Simulation of a Hard-Sphere Fluid in Bicontinuous Random Media

Abstract

The influence of solid-phase connectivity on size-exclusion partitioning and on diffusion of a dilute hard-sphere fluid in overlapping and nonoverlapping spheres models of porous media is investigated using molecular dynamics and Monte Carlo simulation techniques. Four models are examined, two of which are subject to constrained bicontinuity of the pore and solid phases and two in which the solid spheres in the assemblies are randomly distributed in space. It is shown that at moderate to high porosities, connected (bicontinuous) structures lead to a significant increase in the partition and diffusion coefficients when the particles of the pore fluid are of finite size. The consequences of solid phase connectivity are also clearly illustrated in the long-time decay of the velocity autocorrelation function (VACF) of the diffusing particles, particularly in the vicinity of the percolation threshold. Under these conditions the power law exponents on the long-time tail of the VACF are generally found to be higher in connected models than in random systems and the importance of this result is demonstrated using one of the scaling rules of percolation theory. The simulation results are also compared with the predictions of current theories of partitioning and diffusion in random sphere assemblies and, with reference to experimental data available from the literature, it is shown that bicontinuous models are better representations of real porous media.     

Excessive sulphur accumulation and ionic storage behaviour identified in species of Acacia (Leguminosae: Mimosoideae)

Background and Aims Thiophores, which are typically desert gypsophytes, accumulate high (2–6 % S dry weight) sulphur concentrations and may possess unique tolerance to environmental stress factors, e.g. sulphate/metal toxicity, drought and salinity. Little is known of the prevalence of the behaviour or the associated physiological aspects. The aim of this study was to (a) determine the prevalence of thiophore behaviour in a group of Australian xerophytes; (b) identify elemental uptake/storage characteristics of these thiophores; and (c) determine whether the behaviour is constitutive or environmental.Methods The elemental composition of soils and the foliage of 11 species (seven genera) at a site in the Tanami Desert (NT, Australia) was compared and 13 additional Acacia species from other locations were examined for elevated calcium and sulphur concentrations and calcium–sulphur mineralization, thought to be particular to thiophores.Key Results Acacia bivenosa DC. and 11 closely related species were identified as thiophores that can accumulate high levels of sulphur (up to 3·2 %) and calcium (up to 6.8 %), but no thiophores were identified in other genera occupying the same habitat. This behaviour was observed in several populations from diverse habitats, from samples collected over three decades. It was also observed that these thiophores featured gypsum (CaSO₄·2H₂O) crystal druses that completely filled cells and vascular systems in their dried phyllode tissues.Conclusions The thiophores studied exhibit a tight coupling between sulphur and calcium uptake and storage, and apparently store these elements as inorganic salts within the cells of their foliage. Thiophore behaviour is a constitutive trait shared by closely related Acacia but is not highly prevalent within, nor exclusive to, xerophytes. Several of the newly identified thiophores occupy coastal or riparian habitats, suggesting that the evolutionary and ecophysiological explanations for this trait do not lie solely in adaptation to arid conditions or gypsiferous soils.     

Relative importance of factors controlling the leaching and uptake of inorganic ions in the canopy of a spruce forest

Sequential sampling of precipitation under mature spruce trees and time-series analysis of the data were performed in order to assess, in natural conditions, the relative importance of different factors that could influence the leaching and uptake of inorganic ions in the canopy. Eleven rain events were analyzed in order to estimate how external factors, rain intensity, H+, and ionic concentration of the incident rain influence the ionic throughfall concentrations and the net throughfall fluxes. The results led to the conclusion that leaching or uptake mostly occur by diffusion. The influence of the open rain acidity was not conclusive; however, it was shown that the tested external factors only controlled a few percent of the variation of the data. By contrast, the autocorrelation of the data always explained a large portion of the variance. It could result from the gradual changes in the course time of internal factors related to the exchange system including waxes, cuticles, apoplast and xylem sap. These constituents were known to control the exchange at the canopy surface and to be sensitive to the plant physiology and environmental conditions.     

Pore Waters and the Transformation of Nutrients in Marine Subtidal Sands

The contents of ammonia, nitrites, nitrates, phosphates, and silicates were measured in pore waters of subtidal sands in Vostok Bay, the Sea of Japan. The exchange of nutrients between sand and pore waters was studied in the laboratory using a running-water system while percolating the ground with seawater. The composition of pore waters depended on the method and length of time spent sampling, and also on the distance from the ground surface. Statistical analysis did not reveal stable profiles of nutrient concentrations in sand. The velocities of the release and consumption of nutrients in the experiments were rather low and depended on temperature, length of time spent sampling, duration of measurements, and distance from the sand surface. Release and consumption were connected with absorption and desorption, and were not due solely to the biological process of mineralization of organic matter. Analysis of the data obtained is complicated by the canalization of the water flow in the sand and by the consolidation of the sediment during the measurements. The comparison of pore and near-bottom waters makes it possible to hypothesize that the mineralization of organic matter and the release of nutrients into the pore waters occur throughout the whole column of sediment. The mixing of the seawater and groundwater occurs as a result of advection processes, the most important of which is wave percolation.     

Evolution of temperature and salt structure of Lake Bonney, a chemically stratified Antarctic lake

A resurgence of interest in the ecology of perennially ice-covered lakes in the McMurdo dry valleys has necessitated a review of our knowledge of the physical and chemical properties of these unusual lakes. Salinities in the ice-covered lakes cover a range from freshwater to hypersaline brines. Recent measurements of salt composition and concentrations in Lake Bonney reveal little change below the chemocline since extensive measurements made in 1960–1961, although lake level has risen by approximately 5 m since that time. The rise in lake level has resulted in a thickening of the freshwater layer above the chemocline. Temperature structure has adjusted to the effects of increased lake level on heat transfer processes such as transmission and absorption of solar radiation in the water column.Questions about how water-column stability affects biology in Lake Bonney have motivated the formulation of a method to compute density from in situ measurements of temperature, conductivity and pressure. Owing to high salt concentration and unique ion ratios, we modified the UNESCO Equation of State for seawater to predict density at salinities greater than 42. The modifications merge smoothly with the UNESCO equations at a salinity of 42. At salinities below 42 the UNESCO equations give excellent predictions of density.