The metabolic impact of zinc oxide on porcine intestinal cells and enterotoxigenic Escherichia coli K88

Pharmacological levels of zinc oxide (ZnO) incorporated into the post-weaning piglet diet reduce the incidence of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) K88. The mechanism for this is not understood. Here, Intestinal Porcine Epithelial Cells (IPEC) J2 were used as an in vitro model of the porcine intestine. ZnO reduced IPEC J2 viability at concentrations >= 200 mu M, and ETEC adhesion to the host cell was unaffected by ZnO. Characterisation of the metabolism of IPEC J2 cells and ETEC established the effects of ZnO treatment on the metabolic profile of both. Although 100 mu M ZnO did not inhibit growth of either host or pathogen in fully supplemented media, metabolic profiles were significantly altered. Glucose and mannose were essential energy sources for IPEC J2 cells in the presence of ZnO, as the ability to utilise other sources was compromised. The increase in specificity of requirements to support respiration in ETEC was more pronounced, in particular the need for cysteine as a nitrogen source. These findings indicate that ZnO impacts on both host cell and pathogen metabolism and may provide insight into the mechanism for diarrhoea reduction. (C) 2010 Elsevier B.V. All rights reserved.

Field trial using bone meal amendments to remediate mine waste derived soil contaminated with zinc, lead and cadmium

Bone meal amendments are being considered as a remediation method for metal-contaminated wastes. In various forms (biogenic, geogenic or synthetic), apatite, the principal mineral constituent of bone, has shown promise as an amendment to remediate metal-contaminated soils via the formation of insoluble phosphates of Pb and possibly other metals. The efficacy of commercially available bovine bone meal in this role was investigated in a field trial at Nenthead, Cumbria with a mine waste derived soil contaminated with Zn, Pb and Cd. Two 5 m(2) plots were set up: the first as a control and the second, a treatment plot where the soil was thoroughly mixed with bone meal to a depth of 50 cm at a soil to amendment ratio of 25:1 by weight. An array of soil solution samplers (Rhizon SMS (TM)) were installed in both plots and the soil pore water was collected and analysed for Ca, Cd, Zn and Pb regularly over a period of 2 a. Concurrently with the field trial, a laboratory trial with 800 mm high and 100 mm wide leaching Columns Was conducted using identical samplers and with soil from the held site. A substantial release of Zn, Pb, Cd and Ca was observed associated with the bone meal treatment. This release was transient in the case of the leaching columns, and showed seasonal variation in the case of the field trial. It is proposed that this effect resulted from metal complexation with organic acids released during breakdown of the bone meal organic fraction and was facilitated by the relatively high soil pH of 7.6-8.0. Even after this transient release effect had subsided or when incinerated bone meal was substituted in order to eliminate the organic fraction, no detectable decrease in dissolved metals was observed and no P was detected in solution, in contrast with an earlier small column laboratory study. It is concluded that due to the relative insolubility of apatite at above-neutral pH, the rate of supply of phosphate to soil solution was insufficient to result in significant precipitation of metal phosphates and that this may limit the effectiveness of the method to more acidic soils. (c) 2008 Elsevier Ltd. All rights reserved.

Block copolymers of ethylene oxide and phenyl glycidyl ether: micellization, gelation, and drug solubilization

Three triblock copolymers of ethylene oxide and phenyl glycidyl ether, type E(m)G(n)E(m), where G = OCH2-CH(CH2OC6H5) and E = OCH2CH2, were synthesized and characterized by gel-permeation chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and NMR spectroscopy. Their association properties in aqueous solution were investigated by surface tensiometry and light scattering, yielding values of the critical micelle concentration (cmc), the hydrodynamic radius, and the association number. Gel boundaries in concentrated micellar solution were investigated by tube inversion, and for one copolymer, the temperature and frequency dependence of the dynamic moduli served to confirm and extend the phase diagram and to highlight gel properties. Small-angle X-ray scattering was used to investigate gel structure. The overall aim of the work was to define a block copolymer micellar system with better solubilization capacity for poorly soluble aromatic drugs than had been achieved so far by use of block copoly(oxyalkylene)s. Judged by the solubilization of griseofulvin in aqueous solutions of the E(m)G(n)E(m) copolymers, this aim was achieved.

Non-stoichiometric oxide and metal interfaces and reactions

We have employed a combination of experimental surface science techniques and density functional calculations to study the reduction of TiO2(110) surfaces through the doping with submonolayer transition metals. We concentrate on the role of Ti adatoms in self doping of rutile and contrast the behaviour to that of Cr. DFT+U calculations enable identification of probable adsorption structures and their spectroscopic characteristics. Adsorption of both metals leads to a broken symmetry and an asymmetric charge transfer localised around the defect site of a mixed localised/delocalised character. Charge transfer creates defect states with Ti 3d character in the band gap at similar to 1-eV binding energy. Cr adsorption, however, leads to a very large shift in the valence-band edge to higher binding energy and the creation of Cr 3d states at 2.8-eV binding energy. Low-temperature oxidation lifts the Ti-derived band-gap states and modifies the intensity of the Cr features, indicative of a change of oxidation state from Cr3+ to Cr4+. Higher temperature processing leads to a loss of Cr from the surface region, indicative of its substitution into the bulk.

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS (TM) clones with insert sizes similar to 20-40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter beta-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor > 40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

Comparative effect of dairy fatty acids on cell adhesion molecules, nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells

Dairy intake, despite its high saturated fatty acid (SFA) content, is associated with a lower risk of cardiovascular disease and diabetes. This in vitro study determined the effect of individual fatty acids (FA) found in dairy, and FA mixtures representative of a high SFA and a low SFA dairy lipid on markers of endothelial function in healthy and type II diabetic aortic endothelial cells.

Dinuclear copper and zinc complexes of a hexaazamacrocycle containing p-xylyl spacers and bridging anions: theoretical and spectroscopic studies

The hexaazamacrocycle 7,22-dimethyl-3,7,11,18,22,26-hexaazatricyclo[26.2.2.2(13,16)] tetratriaconta-1(30), 13,15,28,31,33- hexaene (Me-2[30] pbz(2)N(6)) was synthesized and characterised by single crystal X-ray diffraction. The macrocycle adopts a conformation with the two aromatic rings almost parallel at a distance of ca. 4.24 Angstrom, but displaced relative to each other by ca. 1.51 Angstrom. The protonation constants of this compound and the stability constants of its complexes with Cu2+ and Zn2+, were determined in water - methanol (9 : 1 v/v) at 25 degreesC with ionic strength 0.10 mol dm(-3) in KCl. The potentiometric and spectroscopic studies (NMR of zinc, cadmium and lead complexes, and EPR of the copper complexes) indicate the formation of only dinuclear complexes. The association constants of the dinuclear copper complex with anions ( thiocyanate, terephthalate and glyphosate) and neutral molecules (1,4-benzenedimethanol, p-xylylenediamine and terephthalic acid) were determined at 20 degreesC in methanol. The structural preferences of this ligand and of its dinuclear copper(II) complex with a variety of bridging ligands were evaluated theoretically by molecular mechanics calculations (MM) and molecular dynamics (MD) using quenching techniques.

Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion

Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

Microwave spectra and centrifugal distortion constants of oxetane

The microwave spectra of oxetane (trimethylene oxide) and its three symmetrically deuterated isotopic species have been observed on a Hewlett-Packard microwave spectrometer from 26.5 to 40 GHz. For the parent species, the β-d2 and the αα′-d4 species, about 300 lines have been assigned for each molecule, and for the d6 species more than 600 lines have been assigned. The assignments range from v = 0 to v = 5 in the puckering vibration; although they are mostly Q transitions, either 3 or 4 R transitions have been observed for each vibrational state. The spectra have been interpreted using an effective rotational hamiltonian for each vibrational state, including five quartic distortion constants according to Watson's formulation, and a variable number of sextic distortion constants; in general, the lines are fitted to about ± 10 kHz. The distortion constants show an anomalous zig-zag dependence on the puckering vibrational quantum number, similar to that first observed for the rotational constants by Gwinn and coworkers. This is interpreted according to a simple modification of the standard theory of centrifugal distortion, involving the double minimum potential function in the puckering coordinate.

Photocatalytic degradation of humic acid in saline waters part 2. Effects of various photocatalytic materials

The present study explores for the first time, the effectiveness of photocatalytic oxidation of. humic acid (HA) in the increasingly important highly saline water. TiO2 (Degussa P25), TiO2 (Anatase), TiO2 (Rutile), TiO2 (Mesoporous) and ZnO dispersions were used as catalysts employing a medium pressure mercury lamp. The effect of platinum loading on P25 and zinc oxide was also investigated. The zinc oxide with 0.3% platinum loading was the most efficient catalyst. The preferred medium for the degradation of HA using ZnO is alkaline, whereas for TiO2 it is acidic. In addition, a comparative study of HA decomposition in artificial seawater (ASW) and natural seawater (NSW) is reported, and the surface areas and band gaps of the catalysts employed were also determined. A spectrophotometric method was used to estimate the extent of degradation of HA. (C) 2003 Elsevier Science B.V. All rights reserved.

Higher oxides of chlorine: absorption cross-sections of Cl2O6 and Cl2O4, the decomposition of Cl2O6, and the reactions of OClO with O and O-3

The absorption cross-sections of Cl2O6 and Cl2O4 have been obtained using a fast flow reactor with a diode array spectrometer (DAS) detection system. The absorption cross-sections at the wavelengths of maximum absorption (lambda(max)) determined in this study are those of Cl2O6: (1.47 +/- 0.15) x 10(-17) cm(2) molecule(-1), at lambda(max) = 276 nm and T = 298 K; and Cl2O4: (9.0 +/- 2.0) x 10(-19) cm(2) molecule(-1), at lambda(max) = 234 nm and T = 298 K. Errors quoted are two standard deviations together with estimates of the systematic error. The shapes of the absorption spectra were obtained over the wavelength range 200-450 nm for Cl2O6 and 200-350 nm for Cl2O4, and were normalized to the absolute cross-sections obtained at lambda(max) for each oxide, and are presented at 1 nm intervals. These data are discussed in relation to previous measurements. The reaction of O with OCIO has been investigated with the objective of observing transient spectroscopic absorptions. A transient absorption was seen, and the possibility is explored of identifying the species with the elusive sym-ClO3 or ClO4, both of which have been characterized in matrices, but not in the gas-phase. The photolysis of OCIO was also re-examined, with emphasis being placed on the products of reaction. UV absorptions attributable to one of the isomers of the ClO dimer, chloryl chloride (ClClO2) were observed; some Cl2O4 was also found at long photolysis times, when much of the ClClO2 had itself been photolysed. We suggest that reports of Cl2O6 formation in previous studies could be a consequence of a mistaken identification. At low temperatures, the photolysis of OCIO leads to the formation of Cl2O3 as a result of the addition of the ClO primary product to OCIO. ClClO2 also appears to be one product of the reaction between O-3 and OCIO, especially when the reaction occurs under explosive conditions. We studied the kinetics of the non-explosive process using a stopped-flow technique, and suggest a value for the room-temperature rate coefficient of (4.6 +/- 0.9) x 10(-19) cm(3) molecule(-1) s(-1) (limit quoted is 2sigma random errors). The photochemical and thermal decomposition of Cl2O6 is described in this paper. For photolysis at k = 254 nm, the removal of Cl2O6 is not accompanied by the build up of any other strong absorber. The implications of the results are either that the photolysis of Cl2O6 produces Cl-2 directly, or that the initial photofragments are converted rapidly to Cl-2. In the thermal decomposition of Cl2O6, Cl2O4 was shown to be a product of reaction, although not necessarily the major one. The kinetics of decomposition were investigated using the stopped-flow technique. At relatively high [OCIO] present in the system, the decay kinetics obeyed a first-order law, with a limiting first-order rate coefficient of 0.002 s(-1). (C) 2004 Elsevier B.V. All rights reserved.

Zinc-histidine complex protects cultured cortical neurons against oxidative stress-induced damage

The levels of zinc in the brain are directly affected by dietary zinc and deficiency has been associated with alcohol withdrawal seizures, excitotoxicity, impaired learning and memory and an accelerated rate of dysfunction in aged brain. Although zinc is essential for a healthy nervous system, high concentrations of zinc are neurotoxic, thus it is important to identify the most effective forms of zinc for treatment of conditions of the central nervous system. Accumulating evidence suggests that zinc-histidine complex (Zn(HiS)(2)) has greater biological potency and enhanced bioavailability compared with other zinc salts and also has antioxidant potential. Therefore, in this study we investigated the ability of zinc-histidine to protect cultured cortical neurons against hydrogen peroxide-induced damage. Pre-treating neurons for 18h with subtoxic concentrations of zinc-histidine (5-25 muM) improved neuronal viability and strongly inhibited hydrogen peroxide-induced (75 muM, 30 min) cell damage as assessed by MTT turnover and morphological analysis 24 It later. Low concentrations of zinc-histidine were more neuroprotective than zinc chloride. There was evidence of an anti-apoptotic mechanism of action as zinc-histidine inhibited hydrogen peroxide-induced caspase-3 activation and c-jun-N-terminal kinase phosphorylation. In summary, zinc supplementation with zinc-histidine protects cultured neurons against oxidative insults and inhibits apoptosis which suggests that zinc-histidine may be beneficial in the treatment of diseases of the CNS associated with zinc deficiency. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS

The first application of high field NMR spectroscopy (800 MHz for 1H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional 1H–1H TOCSY and 1H–13C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Zinc chelatase in human lymphocytes: detection of the enzymatic defect in erythropoietic protoporphyria

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.