Integrated understanding of urban land, groundwater, baseflow and surface-water quality--the City of Birmingham, UK

Integrated understanding of urban land, groundwater (shallow and deep), baseflow and surface-water quality relationships is required for effective urban water-quality management. Chemical quality data from across these media have been collected for the Birmingham (UK) aquifer--River Tame conurbation to assess chemical transport from contaminated land to groundwater to baseflow to surface water. Although metals concentrations were high in soils, low leachability and attenuation caused concentrations in groundwaters and baseflow discharging to surface water to be generally low with only sporadic elevated concentrations attributed to localised point sources. Hydrocarbon VOCs (volatile organic compounds) were similarly absent or at low concentration attributable to their ready natural attenuation. Chlorinated VOCs, however, were widely encountered in groundwater, discharging as baseflow to surface water and impacting surface-water quality. This is attributed to their DNAPL (dense nonaqueous-phase liquid) properties and relative recalcitrance although there was some evidence of biodegradation, albeit insufficient to protect surface water and groundwater abstraction receptors. Some inorganic trends were evident across the various media; nitrate was the most significant quality concern. Generic conclusions are drawn on urban water-quality management and the need for risk-based management strategies to optimise use of urban, sporadically contaminated groundwater in conjunction with surface water highlighted.     

Interstitial Oxygen in Tin-Doped Indium Oxide Transparent Conductors

We report first-principles density functional theory calculations of interstitial oxygen in tin-doped indium oxide (ITO), a transparent conducting oxide. Interstitial oxygen plays a critical role in the defect of ITO because it is by removal of interstitial oxygen that n -type charge carriers are produced. The Frank and Köstlin defect model successfully rationalizes the observed conductivity, Sn-doping, and oxygen partial pressure dependencies of ITO by postulating that tin atoms, which substitute for indium, are clustered with interstitial oxygen. Structural evidence for such a clustering, however, remains ambiguous. Recently published Rietveld refinement results of X-ray and neutron diffraction data found interstitial oxygen to be significantly displaced (0.4 Å) from the ideal fourfold position. Our calculations show that the experimental position is plausible only if interstitial oxygen is clustered with Sn_In defects at any of the three d -type cation sites nearest to the interstitial, thereby providing direct structural confirmation of the Frank and Köstlin defect model.     

Carrier fringe analysis algorithms for three degree of freedom optical probing

In this work, we present a fiber-delivered and fiber-detected, 3-DOF optical probe concept for measuring optical components to be used in conjunction with an optical coordinate measuring machine (OCMM). The optical probe uses a Michelson interferometer to produce carrier fringes and a high density fiber bundle to transmit interferograms that are recorded away from the probe head in a remote imaging system. We compare several different signal FFT processing techniques (parabolic interpolation, windowing, and zero padding) and a single-bin DFT technique to compute and enhance the resolution of the displacement, tip, and tilt of a moving mirror. We simulated varying signal-to-noise ratios and interference fringe contrast ranges to determine the algorithms’ sensitivity to those parameters and compare our simulated values to measured SNR and fringe values. Based on this work, it should be possible to use a carrier fringe algorithm for fiber probing applications if the interferogram can be transmitted through the fiber bundle with sufficient contrast (40%) and SNR (30 dB).     

Structural model of the catalytic domain of an enzyme with cell adhesion activity: human vascular adhesion protein-1 (HVAP-1) D4 domain is an amine oxidase

Human vascular adhesion protein-1 (HVAP-1) is a multifunctional protein having at least two different cellular roles, functioning both as a lymphocyte-endothelial cell adhesion protein and as an enzyme with monoamine oxidase activity. HVAP-1 is a 180 kDa homodimeric glycoprotein consisting of a membrane-spanning domain and three predicted extracellular copper-containing amine oxidase domains. In HVAP-1 the extracellular domains are composed of a large domain D4, containing the active site and forming the interface of the dimer, while the smaller D2 and D3 domains surround the D4 dimer near the entrance to the active site. The structural model of the catalytic D4 domain of HVAP-1 reveals that all components necessary for enzymatic monoamine oxidase activity are indeed present within the HVAP-1 and pinpoints residues that may be key to substrate entry through a channel to the active site and residues likely to be involved in substrate specificity as well as structural features critical to dimer formation. Proper glycosylation is required for the cell adhesion function of HVAP- 1 and the predicted location of the sugar units at the solvent-exposed surface suits this function well.      

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is usedas a thermolabile switch in 'hot start' variations of PCR to minimizenon-specific amplification events. Earlier studies have established thatTP7 binds to the polymerase domain of TaqP, competes with primer templatecomplex for binding and is a potent inhibitor of the polymerase activity ofTaqP. We report crystallographic determination of the structure of an Fabfragment of TP7 and computational docking of the structure with the knownthree-dimensional structure of the enzyme. Our observations stronglysuggest that the origin of inhibitory ability of TP7 is its binding toenzyme residues involved in DNA binding and polymerization mechanism.Although criteria unbiased by extant biochemical data have been used inidentification of a putative solution, the resulting complex offers aneminently plausible structural explanation of biochemical observations. Theresults presented are of general significance for interpretation of dockingexperiments and in design of small molecular inhibitors of TaqP, that arenot structurally similar to substrates, for use in PCR. Structural andfunctional similarities noted among DNA polymerases, and the fact thatseveral DNA polymerases are pharmacological targets, make discovery ofnon-substrate based inhibitors of additional importance.     

Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.     

Testis-like steroidogenesis in the ovotestis of the European mole, Talpa europaea

A gene (AglyA) encoding serine hydroxymethyltransferase of Acinetobacter radioresistens CMC-1 was cloned and sequenced. Nucleotide sequence analysis of AglyA predicted a single open reading frame (ORF) of 1251 bp encoding a 417-amino acid polypeptide. Two putative MetR-like binding sites (5'-TGAAACATGAGCT) and (5'-TGAGCAAAGTTCA), centered at bp -123 and -95 relative to the +1 translation start site were found, which have six out of nine and eight out of nine nucleotides that match to the consensus sequence of Escherichia coli (5'-TGAANNT/ANNTTCA), respectively. The enzyme also showed a high level of homology to other sources of serine hydroxymethyltransferase proteins.     

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.     

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.