Analysis of chain and blood group type and branching pattern of sialylated oligosaccharides by negative ion electrospray tandem mass spectrometry

We previously reported sequence determination of neutral oligosaccharides by negative ion electrospray tandem mass spectrometry on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without the need of derivatization. In the present report, we extend our strategies to sialylated oligosaccharides for analysis of chain and blood group types together with branching patterns. A main feature in the negative ion mass spectrometry approach is the unique double glycosidic cleavage induced by 3-glycosidic substitution, producing characteristic D-type fragments which can be used to distinguish the type 1 and type 2 chains, the blood group related Lewis determinants, 3,6-disubstituted core branching patterns, and to assign the structural details of each of the branches. Twenty mono- and disialylated linear and branched oligosaccharides were used for the investigation, and the sensitivity achieved is in the femtomole range. To demonstrate the efficacy of the strategy, we have determined a novel complex disialylated and monofucosylated tridecasaccharide that is based on the lacto-N-decaose core. The structure and sequence assignment was corroborated by :methylation analysis and H-1 NMR spectroscopy.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

First 5f-element complexes with the tetradentate BTBP ligand. Synthesis and crystal structure of uranyl(VI) compounds with CyMe4BTBP

Treatment of [UO2(OTf)(2)] or [UO2I2(thf)(3)] with 1 equiv. of CyMe4BTBP in anhydrous acetonitrile led to the formation of [UO2(CyMe4BTBP)(OTf)(2)] (1) and [UO2(CyMe4BTBP)I-2] (2) which crystallized as the cationic forms [UO2(CyMe4BTBP)(py)][OTf](2) (3) and [UO2I(CyMe4BTBP)][I] (4) in pyridine and acetonitrile, respectively. These compounds are unique examples of structurally characterized actinide complexes with a BTBP molecule; this ligand adopts a planar conformation in the equatorial plane of the {UO2}(2+) ion. In pyridine, 1 is dissociated into [UO2(OTf)(2)(PY)(3)] and free CyMe4BTBP and the thermodynamic parameters (K, Delta H, Delta S) of this equilibrium have been determined by H-1 NMR spectroscopy. The ethoxide derivative [UO2(OEt)(CyMe4BTBP)][OTf] (5) crystallized from a solution of I in a mixture of ethanol and acetone under air, and the dinuclear mu-oxo complex [{UO2(CyMe4BTBP)}(2)(mu-O)][I](2) (6) was obtained from [UO2I(thf)(2.7)] and CyMe4BTBP. The crystal structures of 6 and of the analogous derivatives [{UO2(py)(4)}(2)(mu-O)][I](2)(7) and [{UO2(TPTZ)(py)}(2)(mu-O)][I-3](2)(8) exhibit a flexible [{UO2}-O-{UO2}](2+) moiety.

Quartz crystal microbalance monitoring of density changes in mesoporous TiO2 phytate films during redox and ion exchange processes

Nanofilm deposits of TiO2 nanoparticle phytates are formed on gold electrode surfaces by 'directed assembly' methods. Alternate exposure of a 3-mercapto-propionic acid modified gold surface to (i) a TiO2 sol and (ii) an aqueous phytic acid solution (pH 3) results in layer-by-layer formation of a mesoporous film. Ru(NH3)(6)(3+) is shown to strongly adsorb/accumulate into the mesoporous structure whilst remaining electrochemically active. Scanning the electrode potential into a sufficiently negative potential range allows the Ru(NH3)(6)(3+) complex to be reduced to Ru(NH3)(6)(2+) which undergoes immediate desorption. When applied to a gold coated quartz crystal microbalance (QCM) sensor, electrochemically driven adsorption and desorption processes in the mesoporous structure become directly detectable as a frequency response, which corresponds directly to a mass or density change in the membrane. The frequency response (at least for thin films) is proportional to the thickness of the mass-responsive film, which suggests good mechanical coupling between electrode and film. Based on this observation, a method for the amplified QCM detection of small mass/density changes is proposed by conducting measurements in rigid mesoporous structures. (C) 2003 Elsevier Science B.V. All rights reserved.

The role of impurities on the low temperature CO oxidation on Au/TiO2

A number of Au/TiO2 catalysts have been prepared by a variety of methods in order to assess the affect of catalysts preparation methods on performance, catalyst contamination and the interplay between them. Their activity was studied in a pulse flow microreactor and it was found that preparation methods designed to eliminate impurities lead to more active samples. The effect of chlorine is often cited to be detrimental in the literature, but we have quantified it using XPS. It was found that the activity decreased in a nearly linear fashion with an increasing amount of this element at the surface. It is generally considered that catalysts prepared by the incipient wetness (IW) technique are ineffective for CO oxidation, but we show here that, by appropriate preparation methods, high activity IW catalysts can be made.

Calcium removal from milk by ion exchange

Calcium removal, using Duolite C433 ion exchange resin, was faster from permeate than from milk. Almost all calcium could be removed, suggesting a fairly rapid conversion from both soluble calcium phosphate and from micellar calcium to ionic calcium. Calcium reduction from milk is accompanied by an increase in pH, a reduction in ionic calcium, an increase in ethanol stability and an increase in the rennet coagulation time. There is a gradual increase in the average casein micelle size with calcium removal, up to a point where the micelle size increases dramatically. Zeta potential becomes more negative with calcium removal. At higher levels of calcium removal, the changes are not reversible, on reducing pH to its original value. For goat's milk, over the range 0-20% total calcium removal, relatively small reductions in total calcium gave rise to proportionally larger reductions in ionic calcium in a ratio of about 1:3.2.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

Cusp ion steps, field-aligned currents and poleward moving auroral forms

We predict the field-aligned currents around cusp ion steps produced by pulsed reconnection between the geomagnetic field and an interplanetary magnetic field (IMF) with a B-Y component that is large in magnitude. For B-Y > 0, patches of newly opened flux move westward and eastward in the Northern and Southern Hemispheres, respectively, under the influence of the magnetic curvature force. These flow directions are reversed for B-Y < 0. The speed of this longitudinal motion initially grows with elapsed time since reconnection, but then decays as the newly opened field lines straighten. We predict sheets of field-aligned current on the boundaries between the patches produced by successive reconnection pulses, associated with the difference in the speeds of their longitudinal motion. For low elapsed times since reconnection, near the equatorward edge of the cusp region where the field lines are accelerating, the field-aligned current sheets will be downward or upward in both hemispheres for positive or negative IMF B-Y, respectively. At larger elapsed times since reconnection, as events slow and evolve from the cusp into the mantle region, these field-aligned current directions will be reversed. Observations by the Polar spacecraft on August 26,1998, show the predicted upward current sheets at steps seen in the mantle region for IMF B-Y > 0. Mapped into the ionosphere, the steps coincide with poleward moving events seen by the CUTLASS HF radar. The mapped location of the largest step also coincides with a poleward moving arc seen by the UVI imager on Polar. We show that the arc is consistent with a region of upward field-aligned current that has become unstable, such that a potential drop of about 1 kV formed below the spacecraft. The importance of these observations is that they confirm that the poleward moving events, as seen by the HF radar and the UV imager, are due to pulsed magnetopause reconnection. Milan et al. [2000] noted that the great longitudinal extent of these events means that the required reconnection pulses would have contributed almost all the voltage placed across the magnetosphere at this time. The observations also show that auroral arcs can form on open field lines in response to the pulsed application of voltage at the magnetopause.

Auroral bright spot sequence near 1400 MLT: Coordinated optical and ion drift observations

Optical observations of a dayside auroral brightening sequence, by means of all-sky TV cameras and meridian scanning photometers, have been combined with EISCAT ion drift observations within the same invariant latitude-MLT sector. The observations were made during a January 1989 campaign by utilizing the high F region ion densities during the maximum phase of the solar cycle. The characteristic intermittent optical events, covering ∼300 km in east-west extent, move eastward (antisunward) along the poleward boundary of the persistent background aurora at velocities of ∼1.5 km s−1 and are associated with ion flows which swing from eastward to westward, with a subsequent return to eastward, during the interval of a few minutes when there is enhanced auroral emission within the radar field of view. The breakup of discrete auroral forms occurs at the reversal (negative potential) that forms between eastward plasma flow, maximizing near the persistent arc poleward boundary, and strong transient westward flow to the south. The reported events, covering a 35 min interval around 1400 MLT, are embedded within a longer period of similar auroral activity between 0830 (1200 MLT) and 1300 UT (1600 MLT). These observations are discussed in relation to recent models of boundary layer plasma dynamics and the associated magnetosphere-ionosphere coupling. The ionospheric events may correspond to large-scale wave like motions of the low-latitude boundary layer (LLBL)/plasma sheet (PS) boundary. On the basis of this interpretation the observed spot size, speed and repetition period (∼10 min) give a wavelength (the distance between spots) of ∼900 km in the present case. The events can also be explained as ionospheric signatures of newly opened flux tubes associated with reconnection bursts at the magnetopause near 1400 MLT. We also discuss these data in relation to random, patchy reconnection (as has recently been invoked to explain the presence of the sheathlike plasma on closed field lines in the LLBL). In view of the lack of IMF data, and the existing uncertainty on the location of the open-closed field line boundary relative to the optical events, an unambiguous discrimination between the different alternatives is not easily obtained.

‘It Was All My Fault’: negative interpretation bias in depressed adolescents

The extent to which cognitive models of development and maintenance of depression apply to adolescents is largely untested, despite the widespread application of Cognitive Behavior Therapy (CBT) for depressed adolescents. Cognitive models suggest that negative cognitions, including interpretation bias, play a role in etiology and maintenance of depression. Given that cognitive development is incomplete by the teenage years and that CBT is not superior to non-cognitive treatments in the treatment of adolescent depression, it is important to test the underlying model. The primary aim of this study was to test the hypothesis that interpretation biases are exhibited by depressed adolescents. Four groups of adolescents were recruited: clinically-referred depressed (n = 27), clinically-referred non-depressed (n = 24), community with elevated depression symptoms (n = 42) and healthy community (n = 150). Participants completed a 20 item ambiguous scenarios questionnaire. Clinically-referred depressed adolescents made significantly more negative interpretations and rated scenarios as less pleasant than all other groups. The results suggest that this element of the cognitive model of depression is applicable to adolescents. Other aspects of the model should be tested so that cognitive treatment can be modified or adapted if necessary.