The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

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Using a recently developed procedure combining isothermal and non-isothermal current measurements space charge trapping and transport in LDPE was successfully studied. Unaged, thermally and electrically aged samples were investigated. The samples were conditioned before each measurement in order to obtain reproducible results. In the non-isothermal measurements appeared a broad peak (40degreesC to 50degreesC) that was possible to decompose into two or three peaks (35, 45 and 65degreesC). At even higher temperature another peak was sometimes present (85degreesC) depending on the prior sample conditioning. The space charge is trapped near the surface in deep traps (maximum depth of approximate to 15 mum). Relaxation times, mobilities and activation energies have been calculated for different charging/discharging conditions. For unaged samples the reproducibility of the results was poor while for the aged polyethylene it was quite good, meaning that aging helps conditioning. In the electrically aged LDPE there is a decrease of conductivity and the broad peak of the non-isothermal spectra shows a slight shift towards higher temperatures when compared with the data found in the thermally aged polymer.

Using a recently developed procedure combining isothermal and non-isothermal current measurements space charge trapping and transport in LDPE was successfully studied. Unaged, thermally and electrically aged samples were investigated. The samples were conditioned before each measurement in order to obtain reproducible results. In the non-isothermal measurements appeared a broad peak (40degreesC to 50degreesC) that was possible to decompose into two or three peaks (35, 45 and 65degreesC). At even higher temperature another peak was sometimes present (85degreesC) depending on the prior sample conditioning. The space charge is trapped near the surface in deep traps (maximum depth of approximate to 15 mum). Relaxation times, mobilities and activation energies have been calculated for different charging/discharging conditions. For unaged samples the reproducibility of the results was poor while for the aged polyethylene it was quite good, meaning that aging helps conditioning. In the electrically aged LDPE there is a decrease of conductivity and the broad peak of the non-isothermal spectra shows a slight shift towards higher temperatures when compared with the data found in the thermally aged polymer.

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Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

The thermal decomposition of 2-azidoacetamide (N3CH2CONH2) has been studied by matrix-isolation infrared spectroscopy and real-time ultraviolet photoelectron spectroscopy. N2, CH2NH, HNCO, CO, NH3, and HCN are observed as high-temperature decomposition products, while at lower temperatures, the novel imine intermediate H2NCOCHNH is observed in the matrix-isolation IR experiments. The identity of this intermediate is confirmed both by ab initio molecular orbital calculations of its IR spectrum and by the temperature dependence and distribution of products in the photoelectron spectroscopy (PES) and IR studies. Mechanisms are proposed for the formation and decomposition of the intermediate consistent both with the observed results and with estimated activation energies based on pathway calculations.

Equilibrium solubility of flurbiprofen, a nonsteroidal antiinflammatory agent, in supercritical carbon dioxide was measured by a static analytical method in the pressure range from (8.0 to 25.0) MPa, at temperatures of (303.0, 313.0, and 323.0) K. The cosolvent effect of ethanol in the solubility of the bioactive compound in supercritical carbon dioxide was investigated at 18 MPa and 313 K. The results obtained have a potential application in supercritical processes for this drug. Experimental solubility data were correlated with an empirical density-based Chrastil model.

[M(CpBz)(CO)3CH3] (M=Mo, 2a, W, 2b; CpBz=C5(CH2Ph)5) have been prepared and reacted with PCl5 and PhI · Cl2. Depending on the metal and on the chlorinating reagent used [Mo(CpBz) ($η$2-COCH3)Cl3], 3, [W(CpBz)Cl4], 4, [Mo(CpBz)(CO)3Cl], 5 and [Mo(CpBz)Cl4], 6 have been obtained. The molecular structures of all compounds are reported and two conformations have been characterised for the benzyl substituents. In complexes 2a, 2b and 5 one phenyl ring bends towards the metals while in 3 and 4 the five phenyls point opposite to the metals. © 2004 Elsevier B.V. All rights reserved.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-Angstrom resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca(2+)binding site of low affinity.

Crystals of cytochrome c peroxidase from Pseudomonas stutzeri were obtained using sodium citrate and PEG 8000 as precipitants. A complete data set was collected to a resolution of 1.6 A under cryogenic conditions using synchrotron radiation at the ESRF. The crystals belong to space group P2(1), with unit-cell parameters a = 69.29, b = 143.31, c = 76.83 A, beta = 100.78 degrees. Four CCP molecules were found in the asymmetric unit, corresponding to a pair of dimers related by local dyads. The crystal packing in the structure shows that the functional dimers can dimerize, as suggested by previous biochemical studies.

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Monitoring systems have traditionally been developed with rigid objectives and functionalities, and tied to specific languages, libraries and run-time environments. There is a need for more flexible monitoring systems which can be easily adapted to distinct requirements. On-line monitoring has been considered as increasingly important for observation and control of a distributed application. In this paper we discuss monitoring interfaces and architectures which support more extensible monitoring and control services. We describe our work on the development of a distributed monitoring infrastructure, and illustrate how it eases the implementation of a complex distributed debugging architecture. We also discuss several issues concerning support for tool interoperability and illustrate how the cooperation among multiple concurrent tools can ease the task of distributed debugging.

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