Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

Oxygen transport in ferrite-based ceramic membranes: Effects of alumina sintering aid

Moderate additions of Al₂O₃ to strontium ferrite-based mixed conductors, such as SrFe_0.7Al_0.3O_3−δ and La_0.2Sr_0.8Fe_0.8Ga_0.2O_3−δ with the composition close to the solid solution formation limits, make it possible to improve ceramics sinterability, to increase oxygen permeability and to decrease thermal expansion. These effects are associated with the segregation of alumina-rich phases, primarily SrAl₂O₄, and the formation of A-site cation-deficient perovskite. The improved properties of the SrFe_0.7Al_0.3O₃-based material were used to fabricate high-quality tubular membranes for methane conversion reactors. Similar enhancement in sinterability is also observed for another promising parent material of mixed-conducting membranes, La_0.5Sr_0.5FeO_3−δ. However, extensive dissolution of Al³⁺ cations in the iron sublattice, creation of A-site vacancies and changing the La:Sr concentration ratio all lead to decreasing ionic transport in La_0.5Sr_0.5FeO_3−δ. As a result, additions of either Al₂O₃ or SrAl₂O₄ have a deteriorating influence on the oxygen permeation fluxes through La_0.5Sr_0.5FeO₃-based ceramics.     

Clinical evaluation of ultra-high-field MRI for three-dimensional visualisation of tumour size in uveal melanoma patients,with direct relevance to treatment planning

Objectives

To assess the tumour dimensions in uveal melanoma patients using 7-T ocular MRI and compare these values with conventional ultrasound imaging to provide improved information for treatment options.

Materials and methods

Ten uveal melanoma patients were examined on a 7-T MRI system using a custom-built eye coil and dedicated 3D scan sequences to minimise eye-motion-induced image artefacts. The maximum tumour prominence was estimated from the three-dimensional images and compared with the standard clinical evaluation from 2D ultrasound images.

Results

The MRI protocols resulted in high-resolution motion-free images of the eye in which the tumour and surrounding tissues could clearly be discriminated. For eight of the ten patients the MR images showed a slightly different value of tumour prominence (average 1.0 mm difference) compared to the ultrasound measurements, which can be attributed to the oblique cuts through the tumour made by the ultrasound. For two of these patients the more accurate results from the MR images changed the treatment plan, with the smaller tumour dimensions making them eligible for eye-preserving therapy.

Conclusion

High-field ocular MRI can yield a more accurate measurement of the tumour dimensions than conventional ultrasound, which can result in significant changes in the prescribed treatment.

     

Hydrothermal synthesis of carbonate-free submicron-sized barium titanate from an amorphous precursor: Synthesis and characterization

In this paper, the amorphous barium titanate precursor was prepared by the peroxo-hydroxide method and post-treated by various drying procedures, such as: room temperature drying, room temperature vacuum drying and vacuum drying at 50 °C. The objective in the latter two treatments was to increase the Ti-O-Ba bonds of the precursor. The post-treated precursors were compared with the untreated (i.e., ‘wet’) precursor. Also, a barium titanate precursor was prepared by an alkoxide route. Afterwards, the precursors were hydrothermally treated at 200 °C in a 10 M NaOH solution. Vacuum drying of the precursor seemingly promoted the formation of Ti-O-Ti bonds in the hydrothermal end-product. The low Ba:Ti ratio (0.66) of the alkoxide-route prepared precursor lead to a multi-phase hydrothermal product with BaTiO₃ as the main phase. In contrast, phase pure BaTiO₃, i.e. without BaCO₃ contamination, was obtained for the precursor which was dried at room temperature. Cube-shaped and highly crystalline BaTiO₃ particles were observed by electron microscopy for the hydrothermally treated peroxo-hydroxide-route prepared precursor.     

Permeability of porous gelcast scaffolds for bone tissue engineering

The permeability of metallic and ceramic open-cell foams prepared by the gelcasting technique was assessed by fitting of Forchheimer’s equation to experimental pressure drop curves. The ceramic composition was based on pure hydroxyapatite, while the metallic composition was based on titanium metal. Experimental Darcian (k ₁) and non-Darcian (k ₂) permeability constants displayed values in the range 0.40–3.24 × 10^?9 m² and 3.11–175.8 × 10^?6 m respectively. Tortuosity was evaluated by gas diffusion experiments and ranged from 1.67 to 3.60, with porosity between 72 and 81% and average hydraulic pore size between 325 and 473 μm. Such features were compared to data reported in the literature for cancellous bones and synthetic scaffolds for bone graft. A detailed discussion concerning the limitations of Darcy’s law for fitting laboratory data and for predicting fluid flow through scaffolds in real biomedical applications is also performed. Pore size was obtained by image analysis and was also derived from permeation-absorption-diffusion experiments. In both cases, values were within the range expected for porous scaffolds applications.     

COD and AOX Removal and Biodegradability Assessment for Fenton and O3/UV Oxidation Processes: A Case Study from a Graphical Industry Wastewater

Due to process stability and excellent effluent quality, the use of membrane processes is rapidly expanding. However, a drawback is the production of concentrates and their proper disposal. In this study, reverse osmosis concentrate was treated by Fenton and O₃/UV oxidation processes. The concentrate contains halogenated compounds, recalcitrant COD and low biodegradability. The removal of halogenated compounds and the enhancement of biodegradability were examined. Comparing the investigated processes, Fenton oxidation resulted in a better mineralization of organic matter; however, O₃/UV oxidation achieved a better enhancement of the biodegradability. Furthermore, similar degradation of halogenated compounds were observed for both oxidation processes.     

Proton conductivity in perovskite type yttrium doped barium hafnate

Yttria doped Ba-hafnates were prepared by solid state route and the proton conductivity of pressed samples of yttria doped barium hafnate was determined with impedance spectroscopy. The results are compared with existing data for cerates and zirconates. It was found that Ba-hafnates show a level of proton conduction that is comparable to that of doped Ba-zirconates at low and slightly higher at high temperatures. This is in agreement with the prediction based on the ionic radius of the tetravalent atom in the perovskite.     

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.