Density fractionation of forest soils: methodological questions and interpretation of incubation results and turnover time in an ecosystem context

Soil organic matter (SOM) is often separated by physical means to simplify a complex matrix into discrete fractions. A frequent approach to isolating two or more fractions is based on differing particle densities and uses a high density liquid such as sodium polytungstate (SPT). Soil density fractions are often interpreted as organic matter pools with different carbon (C) turnover times, ranging from years to decades or centuries, and with different functional roles for C and nutrient dynamics. In this paper, we discuss the development and mechanistic basis of common density-based methods for dividing soil into distinct organic matter fractions. Further, we directly address the potential effects of dispersing soil in a high density salt solution on the recovered fractions and implications for data interpretation. Soil collected from forested sites at H. J. Andrews Experimental Forest, Oregon and Bousson Experimental Forest, Pennsylvania was separated into light and heavy fractions by floatation in a 1.6 g cm⁻³ solution of SPT. Mass balance calculations revealed that between 17% and 26% of the original bulk soil C and N content was mobilized and subsequently discarded during density fractionation for both soils. In some cases, the light isotope was preferentially mobilized during density fractionation. During a year-long incubation, mathematically recombined density fractions respired ∼40% less than the bulk soil at both sites and light fraction (LF) did not always decompose more than the heavy fraction (HF). Residual amounts of tungsten (W) present even in well-rinsed fractions were enough to reduce microbial respiration by 27% compared to the control in a 90-day incubation of O_a material. However, residual W was nearly eliminated by repeated leaching over the year-long incubation, and is not likely the primary cause of the difference in respiration between summed fractions and bulk soil. Light fraction at Bousson, a deciduous site developed on Alfisols, had a radiocarbon-based mean residence time (MRT) of 2.7 or 89 years, depending on the interpretation of the radiocarbon model, while HF was 317 years. In contrast, both density fractions from H. J. Andrews, a coniferous site developed on andic soils, had approximately the same MRT (117 years and 93 years for LF and HF). At H. J. Andrews the organic matter lost during density separation had a short MRT (19 years) and can account for the difference in respired CO₂ between the summed fractions and the bulk soil. Recognition and consideration of the effects of the density separation procedure on the recovered fractions will help prevent misinterpretation and deepen our understanding of the specific role of the recovered organic matter fractions in the ecological context of the soil studied.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the α-chymotrypsin–proflavin interaction

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K _obs, ΔH _obs⁰). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.     

Significant intramyocellular lipid use during prolonged cycling in endurance-trained males as assessed by three different methodologies

Intramyocellular triacylglycerol (IMTG) has been suggested to represent an important substrate source during exercise. In the present study, IMTG utilization during exercise is assessed through the use of various methodologies. In addition, we identified differences in the use of intramyocellular lipids deposited in the immediate subsarcolemmal (SS) area and those stored in the more central region of the fiber. Contemporary stable isotope technology was applied in combination with muscle tissue sampling before and immediately after 3 h of moderate-intensity cycling exercise (62 +/- 2% Vo(2 max)) in eight well-trained male cyclists. Continuous infusions with [U-13C]palmitate and [6,6-(2)H2]glucose were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate whole body IMTG and glycogen use. Both immunohistochemical analyses of oil red O (ORO)-stained muscle cross sections and biochemical triacylglycerol (TG) extraction were performed to assess muscle lipid content. During exercise, plasma FFA, muscle (and/or lipoprotein)-derived TG, plasma glucose, and muscle glycogen oxidation contributed 24 +/- 2, 22 +/- 3, 11 +/- 1, and 43 +/- 3% to total energy expenditure, respectively. In accordance, a significant net decline in muscle lipid content was observed following exercise as assessed by ORO staining (67 +/- 8%) and biochemical TG extraction (49 +/- 8%), and a positive correlation was observed between methods (r = 0.56; P < 0.05). Lipid depots located in the SS area were utilized to a greater extent than the more centrally located depots. This is the first study to show significant use of IMTG as a substrate source during exercise in healthy males via the concurrent implementation of three major methodologies. In addition, this study shows differences in resting subcellular intramyocellular lipid deposit distribution and in the subsequent net use of these deposits during exercise.     

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH. The paper was edited by a native speaker through American Journal Experts (http://www.journalexperts.com).     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.