Mechanism for activation of triosephosphate isomerase by phosphite dianion: the role of a ligand-driven conformational change

The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei brucei results in a small 6-fold decrease in k(cat)/K(m) for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this mutation leads to a 17-fold increase in the second-order rate constant for the TIM-catalyzed proton transfer reaction of the truncated substrate piece [1-(13)C]glycolaldehyde ([1-(13)C]-GA) in D(2)O, a 25-fold increase in the third-order rate constant for the reaction of the substrate pieces GA and phosphite dianion (HPO(3)(2-)), and a 16-fold decrease in K(d) for binding of HPO(3)(2-) to the free enzyme. Most significantly, the mutation also results in an 11-fold decrease in the extent of activation of the enzyme toward turnover of GA by bound HPO(3)(2-). The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(c)) relative to an inactive open form (E(o)). We propose that this is due to the relief, in L232A mutant TIM, of unfavorable steric interactions between the bulky hydrophobic side chain of Leu-232 and the basic carboxylate side chain of Glu-167, the catalytic base, which destabilize E(c) relative to E(o).     

Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg(-1). It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR-p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR-p-CAP and SS-p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.     

Enantioselective synthesis of decalin structures with all-carbon quaternary centers via one-pot sequential Cope/Rauhut–Currier reaction

The first example of a one-pot sequential Cope/Rauhut–Currier reaction is reported and used to make functionalized decalin structures with all-carbon quaternary stereocenters. The substrates for the new sequential reaction are generated through a six-step sequence including an enantioselective Birch reduction-allylation reaction which makes the overall process asymmetric.     

Highly efficient Yb-doped silica fibers prepared by powder sinter technology

We report on the characteristics of an active fiber with core material made by sintering of Yb-doped silica powders as an alternative to a conventional modified chemical vapor deposition (MCVD) technique. This material provides the possibility to design very large and homogenously rare-earth doped active fiber cores. We have determined a fiber background attenuation of 20 dB/km and measured a slope efficiency of 80%. These values are comparable to established fibers made by MCVD technology.     

Trace analysis of benzophenone-derived compounds in surface waters and sediments using solid-phase extraction and microwave-assisted extraction followed by gas chromatography–mass spectrometry

This study describes a procedure for determining eight benzophenone-derived compounds in surface waters and sediments. These include the pharmaceutical ketoprofen, its phototransformation products 3-ethylbenzophenone and 3-acetylbenzophenone, and five benzophenone-type ultraviolet (UV) filters. The proposed analytical method involves the pre-concentration of water samples by solid-phase extraction (SPE) and microwave-assisted extraction (MAE) of sediment samples followed by derivatization and analysis by gas chromatography–mass spectrometry. Different parameters were investigated to achieve optimal method performance. Recoveries of 91 to 96 % from water samples were obtained using HLB Oasis SPE cartridges, whereas MAE of sediments (30 min at 150 °C) gave recoveries of 80 to 99 %. Limits of detection were between 0.1 and 1.9 ng L^?1 for water samples and from 0.1 to 1.4 ng g^?1 for sediment samples. The developed method was applied to environmental samples and revealed the presence of UV filters in the majority of the surface waters with up to 690 ng L^?1 of 2-hydroxy-4-methoxybenzophenone. By contrast, ketoprofen (≤2,900 ng L^?1) and its degradation products (≤320 ng L^?1) were found in only two rivers, both receiving wastewater treatment plant effluents. Sediment analysis revealed benzophenone to be present in concentrations up to 650 ng g^?1, whereas concentrations of other compounds were considerably lower (≤32 ng L^?1). For the first time, quantifiable amounts of two ketoprofen transformation products in the aqueous environment are reported.     

Small Deformation Measurements of Diffuse Objects: Phase Stepping by Frequency Drift of a He-Ne Laser

We describe a simple phase-stepping speckle interferometer with large path difference, in which phase modulation is achieved by thermal frequency drift of the He-Ne light source. This system is easy to set up, requires no specialised components, is immune from the effects of piezo actuator hysteresis, and is capable of producing phase maps in which the noise is less than 1/20λ.Presented at 1996 International Workshop on Interferometry (IWI ‘96), August 27-29, Saitama, Japan.     

Stereospecificity of hydride transfer for the catalytically recycled NAD in CDP-d-glucose 4,6-dehydratase

CDP-D-glucose 4,6-dehydratase (E_od), found in the biosynthetic pathway of 3,6-dideoxysugars, contains a tightly bound NAD⁺ that is recycled during catalysis. The stereochemical preference of the hydride transfer to and from the coenzyme in E_od was determined to be pro-S by analyzing the NAD⁺ produced when the apoenzyme was incubated with stereospecifically labeled NADH and its product, CDP-6-deoxy-D-glycero-L-threo-4-hexulose.     

A process of students and their instructor developing a final closed-book mathematics exam

This article describes a study, from a Canadian technical institute's upgrading mathematics course, where students played a role in developing the final closed-book exam that they sat. The study involved a process where students developed practice exams and solutions keys, students sat each other's practice exams, students evaluated classmates' solutions to the practice exams, and finally the instructor used questions from the practice exams to develop the ‘live’ final exam. Phenomenography is used to analyse interview data and report students' experiences. Through the results, claims are made that students experienced deep approaches to learning and worked as partners in learning, teaching and assessment during the process of developing the final exam with their instructor.     

Molecular dynamics studies of nanoconfined water in clinoptilolite and heulandite zeolites

The complete periodic series of alkali and alkaline earth cation variants (Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) of clinoptilolite (Si : Al=5) and heulandite (Si : Al=3.5) aluminosilicate zeolites are examined by large-scale molecular dynamics utilizing a flexible SPC water and aluminosilicate force field. Calculated hydration enthalpies, radial distribution functions, and ion coordination environments are used to describe the energetic and structural components of extra-framework species while power spectra are used to examine the intermolecular dynamics. These data are correlated to evaluate the impact of ion-zeolite, ion-water, and water-zeolite interactions on the behavior of nanoconfined water. Analysis of the correlated data clearly indicates that the charge density of extra-framework cations appears to have the greatest influence on librational motions, while the anionic charge of the framework (i.e. Si:Al ratios) has a lesser impact.     

Fe(II)/alpha-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

Fe(II)/alpha-ketoglutarate-dependent hydroxylases uniformly possess a double-stranded beta-helix fold with two conserved histidines and one carboxylate coordinating their mononuclear ferrous ions. Oxidative decomposition of the alpha-keto acid is proposed to generate a ferryl-oxo intermediate capable of hydroxylating unactivated carbon atoms in a myriad of substrates. This Perspective focuses on a subgroup of these enzymes that are involved in pyrimidine salvage, purine decomposition, nucleoside and nucleotide hydroxylation, DNA/RNA repair, and chromatin modification. The varied reaction schemes are presented, and selected structural and kinetic information is summarized.