Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry

This article discusses the features of a newly developed matrix-assisted laser desorption/ionization quadrupole/time-of-flight (MALDI-QqTOF) mass spectrometer that is useful in the analysis of phosphorylated peptides. Aliquots of beta-casein, a commonly used phosphorylated protein standard, were digested with trypsin directly on a non-porous polyurethane membrane used as sample support in MALDI-QqTOF mass spectrometry (MS) experiments. Although a complete peptide map was obtained, it was difficult to obtain sequence information for some of the tryptic fragments, in particular T1-2, which bears four phosphate groups and is thus difficult to ionize in positive mode. This article focuses on the sequencing of this particular fragment by comparing MS/MS spectra obtained using different precursor ions. These precursors associated with T1-2 were [M + H](+), [M + H](2+), and [M + H - nH(3)PO(4)](+) ions. Typically, phosphorylated ions showed facile unimolecular losses of phosphoric acid moieties, and produced limited backbone fragmentation. The abundance of [M + H](2+) ions of T1-2 in the full mass spectrum was low relative to that of [M + H](+). [M + H - 4H(3)PO(4)](+) ions as MS/MS precursors underwent backbone fragmentations, with phosphoserine residues transformed into dehydroalanines or serines. Unusual b + 18 u fragments were observed, although only for segments with previously phosphorylated serines. These partly interfered with c-ions, and were noticeable due to overlapping isotopic envelopes. It was possible to establish the sequence of phosphorylated tryptic fragment T1-2 and the location of phosphate groups using the mass of dehydroalanine residues (69 Da) and b + 18 u fragments as markers. All MS and MS/MS spectra obtained with fully phosphorylated beta-casein were compared with spectra acquired with dephosphorylated beta-casein obtained commercially. These comparisons helped assess the spectral differences caused by the presence of phosphate groups. Also, they highlighted the potential usefulness of conducting dephosphorylation directly on the probe prior to MALDI analysis in future studies.     

Analysis of trace metals in water by in-situ sample pre-concentration combined with wavelength dispersive X-ray fluorescence spectroscopy and inductively coupled plasma-optical emission spectroscopy

In this paper an earlier work on the use of poly(acrylamidoxime) cloth for use as a passive monitor for trace metals in water by characterizing the sorption of several metals onto the cloth using a continuous flow chamber is extended. The monitors consists of amidoxime chelating groups covalently bound to the surface of a textile encased in a common 35 mm slide holder. Placement of this device in the water to be sampled resulted in the uptake of heavy metals by the chelating groups. After removal of the monitor from water, the metals were analyzed using wavelength dispersive X-ray fluorescence spectroscopy (WDXRF) as well as by acid extraction followed by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Correlation between the two methods of analysis varied with maximum correlation of 0.99897 observed for Pb and a minimum correlation of 0.16512 for Mg. The order of the distribution coefficients for the seven metals tested was: Pb> or =Cu>Zn>Cd>Mn>Fe>Mg, in agreement with the order of the stability constants for the amidoxime/hydroxamic acid group for the chelation of the same metals at a pH of 5, with the exception of Fe. Field testing of the monitors was also carried out and a comparison made between active sampling of river water and sampling with the monitors. Results indicated that semiquantitative analysis of trace metals in water may be performed using the passive monitors.     

Computer simulations of atmospheric ?erenkov radiation in large cosmic-ray showers

Summary The computations of Protheroe and Turver (1) have been extended to include the Čerenkov light in showers incident at zenith angles up to 60°. We use results from a wide range of scaling-based models to demonstrate the existence of a unique model-independent relationship between a range of observable Čerenkov parameters and the depth of the electron cascade maximum.

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Riassunto Si estendono i calcoli di Protheroe e Turver per includere la luce di Čerenkov in sciami incidenti ad angoli di zenit fino a 60°. Si usano risultati di una vasta gamma di modelli basati sulla variazione di scala per dimostrare l'esistenza di un'unica relazione indipendente dal modello tra una serie di parametri osservabili di Čerenkov e la profondità del massimo della cascata elettronica.

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Резюме Компьюторное моделирование Протеро и Турвера обобщается на черенковское излучение в ливнях, падающих при заметных вплоть до 60°. Мы используем результаты из широкой области моделей для демонстрации существования единственного соотношения, не зависящего от модели, мезду областью наблюдаемых черенковских параметров и глубиной максимума электронного каскада.

     

Spectral beam combining of 2 μm Tm fiber laser systems

Output beams from three independently frequency-stabilized thulium master-oscillator power-amplifier fiber laser systems were spectrally combined using a plane-ruled metal diffraction grating. Two laser channels were frequency-stabilized with guided mode resonance filters and the third was stabilized using a plane-ruled metal diffraction grating. The systems had output wavelengths between 1984 and 2015 nm, each with a spectral width of 100-450 pm and output powers between 40-120 W. The combined beam had powers up to 49 W and was 32% efficient with respect to the launched pump power.     

Volume Bragg grating stabilized spectrally narrow Tm fiber laser

A Tm-doped large mode area (LMA) silica fiber laser has been locked to a stable wavelength of 2,053.9 nm using a volume Bragg grating (VBG). The measured spectral width of the laser output was <300 pm, limited by the spectrometer resolution. Although this laser had modest output (approximately 5W) and slope efficiency (41%), this new approach to spectrally limiting the output of LMA fiber lasers is inherently extendable to kilowatt powers, opening up several applications including high-power pulsed fiber lasers and spectral beam combining. Performance characteristics of the laser compared to one using a dielectric mirror as a high reflector are described, and the results imply low VBG losses.     

Microextraction of volatile organic compounds using the inside needle capillary adsorption trap (INCAT) device

A novel method of solventless extraction has been developed based on a combination of solid phase micro extraction and purge and trap methods. In this technique, a hollow needle with either a short length of GC capillary column placed inside it, or an internal coating of carbon, is used as the preconcentration device. Sampling may be performed on ambient air, on solution, or the solution headspace, by passing the gas or liquid through the device either actively with a syringe, or passively via diffusion. The VOC are sorbed and concentrated onto either the carbon layer, or the liquid stationary phase of the capillary column, within the needle. Placing the needle into a heated GC injection port thermally desorbs the organic compounds directly into the GC without the need for solvent extraction. Results suggest that this procedure provides a rapid and sensitive alternative method to those currently available.     

Fabrication of Au–Cu2O core–shell nanocube heterostructures

Au–Cu₂O core–shell nanocubes with edge dimensions of 15–45 nm have been produced by annealing a copper grid at 300 °C in air in the presence of tetraoctylammonium bromide-stabilized gold nanoparticles. The microstructure of these novel heterostructures, especially the interface between gold nanoparticle core and Cu₂O shell, has been investigated in detail using high-resolution transmission electron microscopy (HRTEM). Chemical analyses have been performed on a single Au–Cu₂O core–shell nanocube using energy dispersive X-ray spectroscopy (EDS) in the scanning transmission electron microscopy (STEM). Based on the experimental results, a growth mechanism is proposed.     

Identification of oxidative post-translational modification of serum albumin in patients with idiopathic pulmonary arterial hypertension and pulmonary hypertension of sickle cell anemia

Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability that might, in part, be related to oxidative stress. Oxidative post-translational modifications of plasma proteins may serve as hallmarks of disease severity and could result in altered protein function and structure. We hypothesized that serum albumin in patients with PH of SCA undergoes oxidative post-translational modification and that this modification may reflect important mediators of disease pathogenesis that are common to both idiopathic pulmonary arterial hypertension (IPAH) and PH of SCA. To explore this hypothesis, we studied albumin purified from the plasma of patients in four subject groups: SCA and PH, SCA steady-state without PH, IPAH, and normal volunteers. Purified albumin was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using MALDI-TOFMS, we identified that an ion corresponding to a malondialdehyde (MDA)-modified albumin peptide was differentially present in patients with IPAH and PH of SCA. These results were confirmed by dot-blotting and Western analysis. We localized the site of MDA modification to albumin residue K159 using LC/MS/MS. Thus, we have identified an MDA modification of serum albumin that appears to be a common link between PH of SCA and IPAH. This finding supports the notion that oxidative stress modulates the pathogenesis of PH of SCA and suggests that this and other post-translational modifications may be important biomarkers of disease.     

Evaluation of an on-target sample preparation system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in conjunction with normal-flow peptide high-performance liquid chromatography for peptide mass fingerprint analyses

Large-scale mass spectrometry (MS)-based proteomic analyses require high-throughput sample preparation techniques due to the increasing numbers of samples that make up a typical proteomics experiment. Moreover, extensive sample pre-treatment steps are necessary prior to MS acquisition for even the most rapid and robust MS-based proteomics methodology, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS followed by peptide mass fingerprinting (PMF) analysis. These include sample purification and fractionation, removal of digestion buffers or solvents, and spotting of sample with matrix onto the MALDI target. These multiple steps of time-consuming sample handling can result in high overall analysis costs and the likelihood of sample contamination and loss. In order to overcome some of these limitations in sample processing, we have investigated the use of a novel, simple, inexpensive 96-well elastomeric array that affixes to a MALDI target to create an on-target 96-well plate that accommodates a high solution volume (ca. 200 microL), thereby enabling the on-target processing of samples for MALDI-TOFMS. We explored several factors that influence MALDI sample preparation: type of matrix, solution volume, solution organic composition, solution drying rates and matrix/analyte co-crystallization methods. We also investigated the use of the 96-well elastomeric device for coupling MALDI-TOFMS analysis directly to high flow rate (1 mL/min) reversed-phase (rp)-HPLC. By developing an optimized, robust sample preparation protocol, we were able to obtain mass spectra with a high signal-to-noise ratio from peptide standards present at the 50-fmol level in large starting volumes of solution. PMF analyses were possible from 1-pmol and 500-fmol protein-digest standards. Coupling the device to high-flow HPLC (750 microL/min) yielded a robust and semi-automated means to obtain enhanced MALDI-TOFMS data at 500 ng of protein digest. These methodologies developed for this simple, on-target, elastomeric device show promise for streamlining the sample preparation process from HPLC to MALDI-MS.