Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)2SO4 precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS–PAGE it was found to be a monomeric protein with molecular mass 72±2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 μM) and highest specificity constant (Vmax/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 μM and Ki=205 μM, respectively).

Low-temperature synthesis of ordered graphite nanofibres using nickel(II)-exchanged zeolites

Ordered graphite nanofibre formation has been observed at exceptionally low temperatures on admission of ethyne to zeolite Y, which had been exchanged with Ni(II). The samples have been characterised by TEM, carbon analysis, and electronic spectroscopy. Formation of the nanofibres requires no hydrogen, and was not observed when cation exchange was carried out at acidic pH. The observed fibres resemble herring-bone nanofibrils, growing from nickel particles, and ca. 90% have diameters in the range 35-40 nm. Similar fibres have also been grown using nickel-exchanged zeolite beta.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor

It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing > 5% hexasaccharides (DP6) exhibited > 90% binding, with EC50 values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP6, was dose dependent, with an EC50 value of 5.10 mg/mL (5.15 mu M MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP5 and/or DP6, Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.

Reducing fouling during UHT treatment of goat's milk

Whole fresh goat's milk was heat treated at 135 degrees C for 4 s using a miniature UHT plant. The temperature of the milk in the preheating and sterilizer sections, and the milk flow rate were monitored to evaluate the overall heat transfer coefficient (OHTC). The decrease in OHTC was used to estimate the extent of fouling. Goat's milk fouled very quickly and run times of the UHT plant were short. The use of sodium hexametaphosphate, trisodium citrate and cation exchange resins to reduce ionic calcium prior to UHT processing, increased the pH and alcohol stability of the milk and markedly increased the run time of the UHT plant.

The influence of organic acids in relation to acid deposition in controlling the acidity of soil and stream waters on a seasonal basis

Much uncertainty still exists regarding the relative importance of organic acids in relation to acid deposition in controlling the acidity of soil and surface waters. This paper contributes to this debate by presenting analysis of seasonal variations in atmospheric deposition, soil solution and stream water chemistry for two UK headwater catchments with contrasting soils. Acid neutralising capacity (ANC), dissolved organic carbon (DOC) concentrations and the Na:Cl ratio of soil and stream waters displayed strong seasonal patterns with little seasonal variation observed in soil water pH. These patterns, plus the strong relationships between ANC, Cl and DOC, suggest that cation exchange and seasonal changes in the production of DOC and seasalt deposition are driving a shift in the proportion of acidity attributable to strong acid anions, from atmospheric deposition, during winter to predominantly organic acids in summer.

Production and dissolution rates of earthworm-secreted calcium carbonate

Earthworms secrete granules of calcium carbonate. These are potentially important in soil biogeochemical cycles and are routinely recorded in archaeological studies of Quaternary soils. Production rates of calcium carbonate granules by the earthworm Lumbricus terrestris L. were determined over 27 days in a range of soils with differing chemical properties (pH, organic matter content, water holding capacity, bulk composition, cation exchange capacity and exchangeable cations). Production rate varied between soils, lay in the range 0–0.043 mmolCaCO3 (0–4.3 mg) earthworm−1 d−1 with an average rate of 8 × 10−3 mmolCaCO3 (0.8 mg) earthworm−1 d−1 and was significantly correlated (r = 0.68, P ≤ 0.01) with soil pH. In a second experiment lasting 315 days earthworms repeatedly (over periods of 39–57 days) produced comparable masses of granules. Converting individual earthworm granule production rates into fluxes expressed on per hectare of land per year basis depends heavily on estimates of earthworm numbers. Using values of 10–20 L. terrestris m−2 suggests a rate of 18– 3139 molCaCO3 ha−1 yr−1. Data obtained from flow-through dissolution experiments suggest that at near neutral pH, granule geometric surface areanormalised dissolution rates are similar to those for other biogenic and inorganic calcium carbonate. Fits of the data to the dissolution relationship r = k(1 − ˝)n where r = dissolution rate, k = a rate constant, ˝ = relative saturation and n = the reaction order gave values of k = 1.72 × 10−10 mol cm−2 s−1 and n = 1.8 for the geometric surface area-normalised rates and k = 3.51 × 10−13 mol cm−2 s−1 and n = 1.8 for the BET surface area-normalised rates. In 196 day leaching column experiments trends in granule dissolution rate referenced to soil chemistry corresponded to predictions made by the SLIM model for dissolution of limestone in soil. If soil solution approaches saturation with respect to calcium carbonate granule dissolution will slow or even stop and granules be preserved indefinitely. Granules have the potential to be a small but significant component of the biogeochemical cycling of C and Ca in soil.

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r(2) = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.

A review of biochars’ potential role in the remediation, revegetation and restoration of contaminated soils

Biochars are biological residues combusted under low oxygen conditions, resulting in a porous, low density carbon rich material. Their large surface areas and cation exchange capacities, determined to a large extent by source materials and pyrolysis temperatures, enables enhanced sorption of both organic and inorganic contaminants to their surfaces, reducing pollutant mobility when amending contaminated soils. Liming effects or release of carbon into soil solution may increase arsenic mobility, whilst low capital but enhanced retention of plant nutrients can restrict revegetation on degraded soils amended only with biochars; the combination of composts, manures and other amendments with biochars could be their most effective deployment to soils requiring stabilisation by revegetation. Specific mechanisms of contaminant-biochar retention and release over time and the environmental impact of biochar amendments on soil organisms remain somewhat unclear but must be investigated to ensure that the management of environmental pollution coincides with ecological sustainability.

Direct Observation of Millisecond to Second Motions in Proteins by Dipolar CODEX NMR Spectroscopy

We present a site-resolved study of stow (ms to s) motions in a protein in the solid (microcrystalline) state performed with the use of a modified version of the centerband-only detection of exchange (CODEX) NMR experiment. CODEX was originally based on measuring changes in molecular orientation by means of the chemical shift anisotropy (CSA) tensor, and in our modification, angular reorientations of internuclear vectors are observed. The experiment was applied to the study of stow (15)N-(1)H motions of the SH3 domain of chicken a-spectrin. The protein was perdeuterated with partial back-exchange of protons at labile sites. This allowed indirect (proton) detection of (15)N nuclei and thus a significant enhancement of sensitivity. The diluted proton system also made negligible proton-driven spin diffusion between (15)N nuclei, which interferes with the molecular exchange (motion) and hampers the acquisition of dynamic parameters. The experiment has shown that approximately half of the peaks in the 2D (15)N-(1)H correlation spectrum exhibit exchange in a different extent. The correlation time of the slow motion for most peaks is 1 to 3 s. This is the first NMR study of the internal dynamics of proteins in the solid state on the millisecond to second time scale with site-specific spectral resolution that provides both time-scale and geometry information about molecular motions.

Geochemistry of natural chromium occurrence in a sandstone aquifer in Bauru Basin, Sao Paulo State, Brazil

Anomalous concentrations of Cr(VI) occur in groundwaters of the Adamantina Aquifer, in a large region in the western state of Sao Paulo, sometimes exceeding the potability limit (0.05 mg L(-1)). To identify the possible geochemical reactions responsible for the occurrence of Cr in groundwater in Urania, borehole rock samples were collected in order to carry out mineralogical and chemical analyses. In addition, multilevel monitoring wells were installed and groundwater samples were analyzed. Analyses of the borehole rock samples show the occurrence of a geochemical anomaly of Cr in the quartzose sandstones (average concentrations of 221 ppm). Chrome-diopside is one of the main minerals contributing to this anomaly, having an average Cr content of 1505 ppm. Sequential extraction experiments indicated weakly adsorbed Cr in the order of 0.54 ppm, and this quantity is enough to provide the Cr concentrations observed in groundwater. Groundwaters from the monitoring wells proved to be stratified, with the highest concentrations of Cr(VI) (0.13 mg L(-1)) being associated with high redox and pH values (over 10) and high concentrations of Na. Geochemical reactions that may explain the release of Cr from the solid phase to groundwater involve the release of Cr(III) from minerals (like chrome-diopside and Cr-Fe hydroxide), followed by oxidation of Cr(III) to Cr(VI), probably related to the reduction of Mn oxides present in the aquifer. Then cation exchange occurs and dissolution of carbonates which increases the pH of groundwater, resulting in the desorption and mobilization of Cr(VI) into groundwater. (C) 2011 Elsevier Ltd. All rights reserved.

Relation between sedimentary framework and hydrogeology in the Guarani Aquifer System in Sao Paulo state, Brazil

This paper presents the results of a new investigation of the Guarani Aquifer System (SAG) in Sao Paulo state. New data were acquired about sedimentary framework, flow pattern, and hydrogeochemistry. The flow direction in the north of the state is towards the southwest and not towards the west as expected previously. This is linked to the absence of SAG outcrop in the northeast of Sao Paulo state. Both the underlying Piramboia Formation and the overlying Botucatu Formation possess high porosity (18.9% and 19.5%, respectively), which was not modified significantly by diagenetic changes. Investigation of sediments confirmed a zone of chalcedony cement close to the SAG outcrop and a zone of calcite cement in the deep confined zone. The main events in the SAG post-sedimentary history were: (1) adhesion of ferrugineous coatings on grains, (2) infiltration of clays in eodiagenetic stage, (3) regeneration of coatings with formation of smectites, (4) authigenic overgrowth of quartz and K-feldspar in advanced eodiagenetic stage, (5) bitumen cementation of Piramboia Formation in mesodiagenetic stage, (6) cementation by calcite in mesodiagenetic and telodiagenetic stages in Piramboia Formation, (7) formation of secondary porosity by dissolution of unstable minerals after appearance of hydraulic gradient and penetration of the meteoric water caused by the uplift of the Serra do Mar coastal range in the Late Cretaceous, (8) authigenesis of kaolinite and amorphous silica in unconfined zone of the SAG and cation exchange coupled with the dissolution of calcite at the transition between unconfined and confined zone, and (9) authigenesis of analcime in the confined SAG zone. The last two processes are still under operation. The deep zone of the SAG comprises an alkaline pH, Na-HCO(3) groundwater type with old water and enriched delta(13)C values (<-3.9), which evolved from a neutral pH, Ca-HCO(3) groundwater type with young water and depleted delta(13)C values (>-18.8) close to the SAG outcrop. This is consistent with a conceptual geochemical model of the SAG, suggesting dissolution of calcite driven by cation exchange, which occurs at a relatively narrow front recently moving downgradient at much slower rate compared to groundwater flow. More depleted values of delta(18)O in the deep confined zone close to the Parana River compared to values of relative recent recharged water indicate recharge occur during a period of cold climate. The SAG is a ""storage-dominated"" type of aquifer which has to be managed properly to avoid its overexploitation. (C) 2011 Elsevier Ltd. All rights reserved.

Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

Characterization of organic matter from composting of different residues by physicochemical and spectroscopic methods

Chemical and spectroscopic methods were used to characterize organic matter transformations during the composting process. Four different residue mixtures were studied: P1 - garden trimmings (GT) only, P2 - GT plus fresh cattle manure, P3 - GT plus orange pomace and P4 - GT plus filter cake. The thermophilic phase was not reached in PI compost, but the P2, P3 and P4 composts showed all three typical process phases. The thermophilic phase and CEC/C ratio stabilized after 90 days, while C/N ratio and the ash content stabilized after 60 days. The increasing E(4)/E(6) ratio indicated oxidation reactions occurring during the process in the material from P2, P3 and P4. The (13)C NMR and FTIR results suggested extraction of both pectin and lignin in the HA-like fraction. The CEC/C ratio, temperature and E(4)/E(6) ratio showed that within 90 days P2, P3 and P4 composts were humified. However, material from P1 did not show characteristics of humified compost. From these data, it is apparent that C/N ratio and ash content are not reliable methods for monitoring the composting process. (C) 2009 Elsevier Ltd. All rights reserved.

Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni2+-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.