Determination of NAD(P)H by A Bioluminescent Enzyme Fiber Optic Probe

Abstract

A fiber optic probe was interfaced to a photon counting system for the determination of nicotinamide adenine dinucleotide reduced [NAD(P)H] by a bioluminescence method. The reagents employed in a bacterial luciferase/flavin mononucleotide /decanal system were optimized. Attempts were made to increase the quantum yield of the system. Dodecanal, tridecanal, and tetradecanal were evaluated as alternative aldehyde reagents for decanal, and hydrogen peroxide was added to the system. Neither attempt increased the quantum yield of the system. However, a relatively low detection limit of 1.6 × 10^?9 M for NAD(P)H was obtained with a linear dynamic range of 3.8 orders of magnitude. These results demonstrate the sensitivity of this instrumentation and assay.     

Nanocrystal formation in gas-atomized amorphous Al85Ni10La5 alloy

An Al₈₅Ni₁₀La₅ amorphous alloy, produced via gas atomization, was selected to study the mechanisms of nanocrystallization induced by thermal exposure. High resolution transmission electron microscopy results indicated the presence of quenched-in Al nuclei in the amorphous matrix of the atomized powder. However, a eutectic-like reaction, which involved the formation of the Al, Al₁₁La₃, and Al₃Ni phases, was recorded in the first crystallization event (263°C) during differential scanning calorimetry continuous heating. Isothermal annealing experiments conducted below 263°C revealed that the formation of single fcc-Al phase occurred at 235°C. At higher temperatures, growth of the Al crystals occurred with formation of intermetallic phases, leading to a eutectic-like transformation behaviour at 263°C. During the first crystallization stage, nanocrystals were developed in the size range of 5 ～ 30 nm. During the second crystallization event (283°C), a bimodal size distribution of nanocrystals was formed with the smaller size in the range of around 10 ～ 30 nm and the larger size around 100 nm. The influence of pre-existing quenched-in Al nuclei on the microstructural evolution in the amorphous Al₈₅Ni₁₀La₅ alloy is discussed and the effect of the microstructural evolution on the hardening behaviour is described in detail.     

Scanning tunneling microscopy and surface simulation of zinc-blende GaN(001) intrinsic 4x reconstruction: linear gallium tetramers?

Scanning tunneling microscopy images confirm electron diffraction studies that the zinc-blende GaN(001)-4x reconstruction consists of rows aligned along [110] with a spacing along [110] of 4a. Dual-bias imaging shows a 180 degree shift of the corrugation maximum position between the profiles of empty and occupied states, in agreement with surface simulations based on the 4 x 1 linear tetramer model of Neugebauer et al. [Phys. Rev. Lett. 80, 3097 (1998)]. Electronic structure calculations predict a surface band gap of 1.1 eV, close to the measured value of 1.14 eV and the previously predicted value (1.2 eV). Despite the successes of this model, high-resolution images reveal an unexpected 3x periodicity (not seen in diffraction) along the [110] row direction, indicating the need for a 4 x 3 model, and putting into question the existence of linear Ga tetramers.     

Effect of age on detection of gaps in speech and nonspeech markers varying in duration and spectral symmetry

Gap detection thresholds for speech and analogous nonspeech stimuli were determined in younger and older adults with clinically normal hearing in the speech range. Gap detection thresholds were larger for older than for younger listeners in all conditions, with the size of the age difference increasing with stimulus complexity. For both ages, gap detection thresholds were far smaller when the markers before and after the gap were the same (spectrally symmetrical) compared to when they were different (spectrally asymmetrical) for both speech and nonspeech stimuli. Moreover, gap detection thresholds were smaller for nonspeech than for speech stimuli when the markers were spectrally symmetrical but the opposite was observed when the markers were spectrally asymmetrical. This pattern of results may reflect the benefit of activating well-learned gap-dependent phonemic contrasts. The stimulus-dependent age effects were interpreted as reflecting the differential effects of age-dependent losses in temporal processing ability on within- and between-channel gap detection.     

High-pressure crystal structure of elastically isotropic CaTiO3 perovskite under hydrostatic and non-hydrostatic conditions

The structural evolution of orthorhombic CaTiO3 perovskite has been studied using high-pressure single-crystal x-ray diffraction under hydrostatic conditions up to 8.1 GPa and under a non-hydrostatic stress field formed in a diamond anvil cell (DAC) up to 4.7 GPa. Under hydrostatic conditions, the TiO6 octahedra become more tilted and distorted with increasing pressure, similar to other 2:4 perovskites. Under non-hydrostatic conditions, the experiments do not show any apparent difference in the internal structural variation from hydrostatic conditions and no additional tilts and distortions in the TiO6 octahedra are observed, even though the lattice itself becomes distorted due to the non-hydrostatic stress. The similarity between the hydrostatic and non-hydrostatic cases can be ascribed to the fact that CaTiO3 perovskite is nearly elastically isotropic and, as a consequence, its deviatoric unit-cell volume strain produced by the non-hydrostatic stress is very small; in other words, the additional octahedral tilts relevant to the extra unit-cell volume associated with the deviatoric unit-cell volume strain may be totally neglected. This study further addresses the role that three factors--the elastic properties, the crystal orientation and the pressure medium--have on the structural evolution of an orthorhombic perovskite loaded in a DAC under non-hydrostatic conditions. The influence of these factors can be clearly visualized by plotting the three-dimensional distribution of the deviatoric unit-cell volume strain in relation to the cylindrical axis of the DAC and indicates that, if the elasticity of a perovskite is nearly isotropic as it is for CaTiO3, the other two factors become relatively insignificant.     

L1-regularisation for ill-posed problems in variational data assimilation

We consider four-dimensional variational data assimilation (4DVar) and show that it can be interpreted as Tikhonov or L₂-regularisation, a widely used method for solving ill-posed inverse problems. It is known from image restoration and geophysical problems that an alternative regularisation, namely L₁-norm regularisation, recovers sharp edges better than L₂-norm regularisation. We apply this idea to 4DVar for problems where shocks and model error are present and give two examples which show that L₁-norm regularisation performs much better than the standard L₂-norm regularisation in 4DVar. (© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)     

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling,ligand docking,and molecular dynamics results validated by experimental studies

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT₂ G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ～75% transmembrane (TM) sequence identity. Agonists for 5-HT_2C receptors are under development for psychoses; whereas, at 5-HT_2A receptors, antipsychotic effects are associated with antagonists – in fact, 5-HT_2A agonists can cause hallucinations and 5-HT_2B agonists cause cardiotoxicity. It is known that 5-HT_2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT_2C receptor have not been reported. To predict and validate molecular determinants for 5-HT_2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT_2C receptors.     

Separation of fluorescent phosphatidyl inositol phosphates by CE

Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.