Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Iridoid glycosides ofChelone glabra (Scrophulariaceae) and their sequestration by larvae of a sawfly,Tenthredo grandis (Tenthredinidae)

Analysis ofChelone glabra (Scrophulariaceae) by gas chromatography showed that leaves of this plant contained primarily the iridoid glycoside catalpol, and in a few individuals some aucubin was also detected. There was no difference in the iridoid glycoside content of damaged compared to undamaged plants, nor was there a difference between plants collected from a population in Leverett, Massachusetts, and those from plants in an experimental garden in Cambridge, Massachusetts. Larvae and prepupae of the sawfly,Tenthredo grandis (Tenthredinidae) contained catalpol sequestered from the larval host plant. The exuvia also contained catalpol, whereas the frass contained only aucubin. These results indicate that larvae of this sawfly selectively sequester catalpol, eliminating the aucubin in the frass.     

Electrochemical studies of kerosene-pyrolysedcarbon films

Polycrystalline thin films of conducting carbon are deposited on alumina substrates by the pyrolysis of kerosene vapour at 1000C for 2h in argon atmosphere. Preliminary structural analysis is done by XRD, laser-Raman, FTIR and SEM studies. The electrochemical behaviour of as-grown conducting carbon films was investigated in various electrolytes at different pH and the performance was compared with that of platinum and glassy electrodes. The electrochemical window of the kerosene carbon electrode in 100mm H2SO4 was found to be 2.91V which is greater than that of glassy carbon (2.79V) and platinum (2.02V). Cyclic voltammetry reveals that Pt electrode has almost an equal tendency towards hydrogen and oxygen evolution, whereas glassy carbon favours hydrogen evolution and kerosene carbon favours oxygen evolution. It is suggested that the kerosene carbon electrode can be used as an oxygen electrode more efficiently. Unlike diamond films or glassy electrodes, kerosene carbon thin films are of low cost and good stability; they are also easy to grow on various ceramic substrates of any size. Moreover, these electrodes are very economical and promising for application in chlor-alkali industry.     

STABILITY ANALYSIS FOR THE PREDICTION OF THE MINIMUM TEMPERATURE OF FILM BOILING

The stability of film boiling on a vertical, constant temperature wall is analyzed by a steady-state small perturbation method. It was found that large amplifications of the perturbations in the downstream direction are predicted when the vapor Reynolds number falls below a critical value, indicating a great tendency to collapse the vapor film. Using a relatively simple model for the film thickness, the minimum wall temperature required to sustain the vapor film is calculated. The results show a tendency which is in agreement with experimental observation. It is suggested, however, that using a more accurate model for the film thickness can lead to a good prediction of the minimum wall temperature and explain the large effect of liquid subcooling, system pressure, surface tension and other fluid properties.     

Synthesis and structure-activity relationship of the isoindolinyl benzisoxazolpiperidines as potent,selective, and orally active human dopamine D4 receptor antagonists

A new class of potent dopamine D(4) antagonists was discovered with selectivity over dopamine D(2) and the alpha-1 adrenoceptor. The lead compound was discovered by screening our compound collection. The structure-activity relationships of substituted isoindoline rings and the chirality about the hydroxymethyl side chain were explored. The isoindoline analogues showed modest differences in potency and selectivity. The S enantiomer proved to be the more potent enantiomer at the D(4) receptor. Several analogues with greater than 100-fold selectivity for D(4) over D(2) and the alpha-1 adrenoreceptor were discovered. Several selective analogues were active in vivo upon oral or intraperitoneal administration. A chiral synthesis starting from either D- or L-O-benzylserine is also described.     

Anaerobic biological treatment of phenolic wastewater at 15-18 degrees C

Low-temperature, or psychrophilic (<20 degrees C) anaerobic digestion has been proven feasible for the mineralisation of simple wastewaters. In this study, hybrid expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors were used to evaluate the feasibility of psychrophilic digestion for the treatment of phenol-containing wastewater. Efficient chemical oxygen demand and phenol removal were observed at organic and phenol loading rates of 5 kg COD m(-3)d(-1) and 0.4-1.2 kg phenol m(-3)d(-1) (400-1200 mg phenol [l wastewater](-1)), respectively. There was no long-term accumulation of volatile fatty acids in the reactor systems. Methanogenic activity was developed under psychrophilic conditions but anaerobic methane-producing populations remained mesophilic throughout the trial of 415 days.     

Experimental study of early-age behavior of high performance concrete deck slabs under different curing methods

Curing techniques and curing duration have crucial effects to the strength and durability of concrete. Proper curing can protect against moisture loss from fresh concrete. The objective of this experimental study is to examine the early-age behavior of high-performance concrete (HPC) under various curing methods. Laboratory experiments were conducted to investigate the early-age shrinkage development, temperature change, and evaporation rate when different curing methods were used. Four curing techniques and two curing durations were applied to concrete deck slab and cylindrical specimens. The measured experiment data were also compared with several shrinkage prediction models. The results show that proper moisture-curing methods can effectively reduce concrete temperature due to hydration heat and limit the development of early-age shrinkage strains. The concrete of a longer curing duration would yield lower shrinkage deformation and lower evaporation rate.     

Magnetic force microscopy of superparamagnetic nanoparticles

The use of magnetic force microscopy (MFM) to detect probe-sample interactions from superparamagnetic nanoparticles in vitro in ambient atmospheric conditions is reported here. By using both magnetic and nonmagnetic probes in dynamic lift-mode imaging and by controlling the direction and magnitude of the external magnetic field applied to the samples, it is possible to detect and identify the presence of superparamagnetic nanoparticles. The experimental results shown here are in agreement with the estimated sensitivity of the MFM technique. The potential and challenges for localizing nanoscale magnetic domains in biological samples is discussed.