Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Changes in the tear proteins of diabetic patients

BACKGROUND: Previous studies have shown a significant increase in tear protein peaks in the tears of diabetic patients suffering from dry eye. The aim of this study was to analyze the tear protein patterns from patients with diabetes mellitus who do not suffer from ocular surface diseases (DIA). METHODS: A total of 515 patients were examined in this study (255 healthy subjects (controls) and 260 patients suffering from diabetes mellitus). Tear proteins were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. After digital image analysis densitometric data files were created and subsequently used for multivariate statistical procedures. RESULTS: A significant increase in the number of peaks was detected in diabetic patients compared to controls (P < 0.0003). The analysis of discriminance revealed a highly significant discrimination between diabetic patients and controls (Wilks lambda: 0.27; P < 0.000001). Furthermore, a significant difference in the protein pattern of diabetic patients could be detected between those suffering from dry eye or not (P < 0.002). The changes in protein patterns of diabetic patients increased with the duration of the diabetic disease. In diabetic patients with a disease duration longer than 10 years the changes were significantly more expressed than in patients with a shorter diabetic history (P < 0.003) and in healthy subjects (P < 0.0001). CONCLUSIONS: The tear protein patterns of diabetic patients are very different in the number and intensity of spots from those of healthy subjects. Furthermore, it could be demonstrated that the differences found in the tear patterns of diabetic patients are not equal to those found in previous studies in patients suffering from dry-eye disease. The alterations in the diabetic tears were correlated with the duration of the diabetic disease. With longer disease, history changes in the tear protein patterns increased. With the course of the disease some protein peaks appeared that are not present in healthy persons. Our study shows that the analysis of electrophoretic tear protein patterns is a new non-invasive approach in the early diagnosis and analysis of the pathogenesis of diabetes induced ocular surface disease

Interrogation of caesium atoms in a fountain clock by a femtosecond laser microwave oscillator

A caesium fountain clock is operated utilizing a microwave oscillator that derives its frequency stability from a stable laser by means of a fiber-laser femtosecond frequency comb. This oscillator is based on the technology developed for optical clocks and replaces the quartz based microwave oscillator commonly used in fountain clocks. As a result, a significant decrease of the frequency instability of the fountain clock is obtained, reaching 0.74E-14 at 100 s averaging time. We could demonstrate that for a significant range of detected atom numbers the instability is limited by quantum projection noise only, and that for the current status of this fountain clock the new microwave source poses no limit on the achievable frequency instability.Comment: 4 pages, 4 figure

A re-analysis of the three-year WMAP temperature power spectrum and likelihood

We analyze the three-year WMAP temperature anisotropy data seeking to confirm the power spectrum and likelihoods published by the WMAP team. We apply five independent implementations of four algorithms to the power spectrum estimation and two implementations to the parameter estimation. Our single most important result is that we broadly confirm the WMAP power spectrum and analysis. Still, we do find two small but potentially important discrepancies: On large angular scales there is a small power excess in the WMAP spectrum (5-10% at l<~30) primarily due to likelihood approximation issues between 13 <= l <~30. On small angular scales there is a systematic difference between the V- and W-band spectra (few percent at l>~300). Recently, the latter discrepancy was explained by Huffenberger et al. (2006) in terms of over-subtraction of unresolved point sources. As far as the low-l bias is concerned, most parameters are affected by a few tenths of a sigma. The most important effect is seen in n_s. For the combination of WMAP, Acbar and BOOMERanG, the significance of n_s =/ 1 drops from ~2.7 sigma to ~2.3 sigma when correcting for this bias. We propose a few simple improvements to the low-l WMAP likelihood code, and introduce two important extensions to the Gibbs sampling method that allows for proper sampling of the low signal-to-noise regime. Finally, we make the products from the Gibbs sampling analysis publically available, thereby providing a fast and simple route to the exact likelihood without the need of expensive matrix inversions.Comment: 14 pages, 7 figures. Accepted for publication in ApJ. Numerical results unchanged, but interpretation sharpened: Likelihood approximation issues at l=13-30 far more important than potential foreground issues at l <= 12. Gibbs products (spectrum and sky samples, and "easy-to-use" likelihood module) available from http://www.astro.uio.no/~hke/ under "Research

Emplacement and high-temperature evolution of gabbros of the 16.5°N oceanic core complexes (Mid-Atlantic Ridge): Insights ito the compositional variability of the lower oceanic crust

This study reports the composition of the oceanic crust from the 16.5°N region of the Mid‐Atlantic Ridge, a spreading ridge segment characterized by a complex detachment fault system and three main oceanic core complexes (southern, central, and northern OCCs). Lithologies recovered from the core complexes include both greenschist facies and weathered pillow basalt, diabase, peridotite, and gabbro, while only weathered and fresh pillow basalt was dredged from the rift valley floor. The gabbros are compositionally bimodal, with the magmatic crust in the region formed by scattered intrusions of chemically primitive plutonic rocks (i.e., dunites and troctolites), associated with evolved oxide‐bearing gabbros. We use thermodynamic models to infer that this distribution is expected in regions where small gabbroic bodies are intruded into mantle peridotites. The occurrence of ephemeral magma chambers located in the lithospheric mantle enables large proportions of the melt to be erupted after relatively low degrees of fractionation. A large proportion of the dredged gabbros reveal evidence for deformation at high‐temperature conditions. In particular, chemical changes in response to deformation and the occurrence of very high‐temperature ultramylonites (>1000 °C) suggest that the deformation related to the oceanic detachment commenced at near‐solidus conditions. This event was likely associated with the expulsion of interstitial, evolved magmas from the crystal mush, a mechanism that enhanced the formation of disconnected oxide‐gabbro seams or layers often associated with crystal‐plastic fabrics in the host gabbros. This granulite‐grade event was soon followed by hydrothermal alteration revealed by the formation of amphibole‐rich veins at high‐temperature conditions (~900 °C)

Assessment of the use of partitioning and interfacial tracers to determine the content and mass removal rates of nonaqueous phase liquids

It was assessed whether partitioning and interfacial tracers can be used to determine the content and mass removal rate of nonaqueous phase liquid (NAPL) in porous media. Retardation factors for these tracers were determined for five different model matrices contaminated with hexadecane as NAPL. The retardation of the partitioning tracer 2,4-dimethyl-3-pentanol was correlated with the degree of NAPL saturation for four of the five matrices (r(2) = 0.93, n = 8), The observed retardation factors matched the retardation factors predicted with the independently determined hexadecane-water partitioning constant and the degree of NAPL saturation, indicating that this tracer may be used to estimate the degree of NAPL saturation of porous media. The mass removal rates of NAPL from columns packed with different matrices were determined by measuring the amount of hexadecane in the column effluent during elution with electrolyte solution. These removal rates differed over 3 orders of magnitude, dependent on the matrix used. The retardation of the interfacial tracer alkylbenzenesulfonate was higher for matrices with higher NAPL mass removal rates but was not correlated to the degree of NAPL saturation. This indicates that the retardation factors of alkylbenzenesulfonates in NAPL-contaminated media contain information related to the NAPL mass removal rates

Resonances in rotationally inelastic scattering of OH(X2ΠX^2\Pi) with helium and neon

We present detailed calculations on resonances in rotationally and spin-orbit inelastic scattering of OH (X\,^2\Pi, j=3/2, F_1, f) radicals with He and Ne atoms. We calculate new \emph{ab initio} potential energy surfaces for OH-He, and the cross sections derived from these surfaces compare favorably with the recent crossed beam scattering experiment of Kirste \emph{et al.} [Phys. Rev. A \textbf{82}, 042717 (2010)]. We identify both shape and Feshbach resonances in the integral and differential state-to-state scattering cross sections, and we discuss the prospects for experimentally observing scattering resonances using Stark decelerated beams of OH radicals.Comment: 14 pages, 15 Figure

Blending of nanoscale and microscale in uniform large-area sculptured thin-film architectures

The combination of large thickness ($>3$ $\mu$m), large--area uniformity (75 mm diameter), high growth rate (up to 0.4 $\mu$m/min) in assemblies of complex--shaped nanowires on lithographically defined patterns has been achieved for the first time. The nanoscale and the microscale have thus been blended together in sculptured thin films with transverse architectures. SiO$_x$ ($x\approx 2$) nanowires were grown by electron--beam evaporation onto silicon substrates both with and without photoresist lines (1--D arrays) and checkerboard (2--D arrays) patterns. Atomic self--shadowing due to oblique--angle deposition enables the nanowires to grow continuously, to change direction abruptly, and to maintain constant cross--sectional diameter. The selective growth of nanowire assemblies on the top surfaces of both 1--D and 2--D arrays can be understood and predicted using simple geometrical shadowing equations.Comment: 17 pages, 9 figure

Small Is Beautiful: Why Profundaplasty Should Not Be Forgotten

Background: Surgical profundaplasty (SP)is used mainly as an adjunct to endovascular management of peripheral vascular disease (PAD) today. Results from earlier series of profundaplasty alone have been controversial, especially regarding its hemodynamic effect. The question is: Can profundaplasty alone still be useful? Our aim was to evaluate its role in the modern management of vascular patients. Methods: This was a retrospective outcome study. A consecutive series of 97 patients (106 legs) from January 2000 through December 2003 were included. In 55 (52%) legs, the superficial femoral artery was occluded. These patients were included in the current analysis. Of these patients 14 (25%) were female. Mean age was 71 ((11) years. Nineteen (35%) were diabetic. The indication for operation was claudication in 29 (53%), critical leg ischemia (CLI) in 26 (47%), either with rest pain in 17 (31%), or ulcer/gangrene in 9 (16%). Endarterectomy with patch angioplasty with bovine pericardium was performed in all cases. Mean follow-up was 33(14 months. Mean preoperative ankle brachial index (ABI) was 0.6. Sustained clinical efficacy was defined as upward shift of 1 or greater on the Rutherford scale without repeat target limb revascularization (TLR) or amputation. Mortality, morbidity, need for TLR, or amputation were separate endpoints. Results: Postoperatively, ABI was significantly improved (mean=0.7), in 24 (44%) by more than 0.15. At three years, cumulative clinical success rate was 80%. Overall, patients with claudication had a better outcome than those with CLI (p=0.04). Two (4%) major amputations and 2 (4%) minor ones were performed, all in patients with CLI. None of the 9 (16%) ulcers healed. Conclusion: Profundaplasty is still a valuable option for patients with femoral PAD and claudication without tissue loss. It is a straightforward procedure that combines good efficacy with low complication rates. Further endovascular treatment may be facilitated. It is not useful for patients with the combination of critical ischemia and tissue los

Electron localization by a magnetic vortex

We study the problem of an electron in two dimensions in the presence of a magnetic vortex with a step-like profile. Dependending on the values of the effective mass and gyromagnetic factor of the electron, it may be trapped by the vortex. The bound state spectrum is obtained numerically, and some limiting cases are treated analytically.Comment: 8 pages, latex, 4 figure