General acid/base catalysis in the active site of Escherichia coli thioredoxin

Enzymic catalysts of thiol:disulfide oxidoreduction contain two cysteine residues in their active sites. Another common residue is an aspartate (or glutamate), the role of which has been unclear. Escherichia coli thioredoxin (Trx) is the best characterized thiol:disulfide oxidoreductase, and in Trx these three active-site residues are Cys32, Cys35, and Asp26. Structural analyses had indicated that the carboxylate of Asp26 is positioned properly for the deprotonation of the thiol of Cys35, which would facilitate its attack on Cys32 in enzyme-substrate mixed disulfides. Here, Asp26 of Trx was replaced with isologous asparagine and leucine residues. D26N Trx and D26L Trx are reduced and oxidized more slowly than is wild-type Trx during catalysis by E.coli thioredoxin reductase. Stopped-flow spectroscopy demonstrated that the cleavage of the mixed disulfide between Trx and a substrate is slower in the D26N and D26L enzymes. Buffers increase the rate of mixed disulfide cleavage in these variants but not in wild-type Trx. These results indicate that Asp26 serves as an acid/base in the oxidation/reduction reactions catalyzed by Trx. Specifically, Asp26 protonates (during substrate oxidation) or deprotonates (during substrate reduction) the thiol of Cys35. A similar role is likely filled by the analogous aspartate (or glutamate) residue in protein disulfide isomerase, DsbA, and other thiol:disulfide oxidoreductases. Moreover, these results provide the first evidence for general acid/base catalysis in a thiol:disulfide interchange reaction.     

Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells

The seasonal variation in the nutrient composition of Enhydra fluctuans and Marsilea quadrifolia, two edible semi-aquatic plants, was studied in order to promote their consumption as green leafy vegetables. Both plants had a high crude protein content throughout all harvesting seasons. Enhydra fluctuans had a low ash content and was a good source of beta-carotene (3.7 to 4.2 mg/100 g on a fresh weight basis). Marsilea quadrifolia exhibited wide fluctuations between seasons and was not very promising in nutrient composition when compared to other commonly used green leafy vegetables.     

On the modelling of scalar and mass transport in combustor flows

The results of a numerical study of swirling and non-swirling combustor flows with and without density variations are presented. Constant-density arguments are used to justify closure assumptions invoked for the transport equations for turbulent momentum and scalar fluxes, which are written in terms of density-weighted variables. Comparisons are carried out with measurements obtained from three different axisymmetric model combustors. The three experiments cover recirculating flow, swirling flow and variable-density, swirling flow inside model combustors. Together, they offer wide ranging flow conditions to test the validity of the models. Results show that the Reynolds stress/flux models do a credible job of predicting constant-density, swirling and non-swirling combustor flows with passive scalar transport. However, their improvements over algebraic stress/flux models are marginal. The extension of the constant-density models to variable-density flow calculations shows that the models are equally valid for such flows. Therefore, the present results argue well for the adoption of constant-density models for variable-density flows until a successfully validated variable-density model is available.     

Effects of brefeldin-A on Golgi morphology in human cultured fibroblasts observed in three-dimensional stereo scanning electron microscopy

Brefeldin A (BFA) has been reported to cause disassembly of the Golgi. We have used three-dimensional (3-D) high-resolution scanning electron microscopy (HRSEM) to investigate these effects in human skin fibroblast cells. The spontaneous reassembly during prolonged exposure to BFA and some effects of forskolin were observed. A BFA concentration of 5μg/ml caused Golgi complexes to become vesicular, resulting in a progressive decrease in the size of the Golgi. Morphologic changes were visible within 2 min of BFA incubation, and by 30 min no identifiable Golgi could be found. Spontaneous reassembly of the Golgi apparatus upon the removal of the BFA or with continued long-term exposure with BFA could not be confirmed. Preliminary experiments with forskolin were not effective in reversing or inhibiting the effects of BFA in human fibroblast cells grown in culture. This inability for spontaneous reassembly and nonreversal by forskolin may reflect a differential effect of BFA in various cell types. HRSEM has proven to be useful for observing 3-D morphologic effects of BFA in Golgi.     

A PET study of the neural systems of stuttering

The cause of stuttering is unknown. Failure to develop left-hemispheric dominance for speech is a long-standing theory although others implicated the motor system more broadly, often postulating hyperactivity of the right (language nondominant) cerebral hemisphere. As knowledge of motor circuitry has advanced, theories of stuttering have become more anatomically specific, postulating hyperactivity of premotor cortex, either directly or through connectivity with the thalamus and basal ganglia. Alternative theories target the auditory and speech production systems. By contrasting stuttering with fluent speech using positron emission tomography combined with chorus reading to induce fluency, we found support for each of these hypotheses. Stuttering induced widespread overactivations of the motor system in both cerebrum and cerebellum, with right cerebral dominance. Stuttered reading lacked left-lateralized activations of the auditory system, which are thought to support the self-monitoring of speech, and selectively deactivated a frontal-temporal system implicated in speech production. Induced fluency decreased or eliminated the overactivity in most motor areas, and largely reversed the auditory-system underactivations and the deactivation of the speech production system. Thus stuttering is a disorder affecting the multiple neural systems used for speaking.