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Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe3+/Fe2+ couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and IT or oxyferrous KatG.

The partly-articulated postcranial remains of two sauropod skeletons recently found in Tang Vay (Savannakhet Province, Laos) are assigned to the species Tangvayosaurus hoffeti (nov. gen., nov. sp.). The derived characters present in the new material confirm the presence of titanosaurs in South East Asia at the end of the Early Cretaceous, but are not consistent with its placement within Titanosaurus genus as first done by Hoffet in 1942. All of the material relative to this species is therefore referred to a new genus: Tangvayosaurus. Tangvayosaurus and the Thai genus Phuwiangosaurus have strong affinities and are considered as primitive titanosaurs.

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Tetraheme cytochrome c(3) (13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic, in vitro, part of the electron-transfer chain that operates between substract electron donors and respiratory electron accepters partners in Desulfovibrio species (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994) Biochemistry 33, 6394-6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988) Biochemistry 27, 2444-2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989) fur. J. Biochem. 185, 695-700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide {[}EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins. (C) 1999 Academic Press.

Science and technology (S&T) are considered to be a central source, or at least a basic medium, of societal and industrial innovation, while innovation is conceived to basically feed the regeneration of our welfare. The suppliers of S&T in Europe as well as the users of their „products“, are confronted with a number of challenges today. We want to stress here that it was not the primary goal of our Advanced Science & Technology Policy Planning (ASTPP) Network to come up with proposals how the strategic character of European S&T policies could be strengthened. The ASTPP-network instead focuses on one aspect: the provision of strategic intelligence necessary to identify and develop strategic choices. The underlying hypothesis is that the existing body of experiences with technology foresight, technology assessment and S/T policy evaluation provides a basis for the development of an advanced S&T policy „planning“ approach by trying to enhance, interlink or even integrate the growing, but still dispersed experience in these three areas of intelligence. By „intelligent“ we mean that the inter-relatedness of S&T, industrial efforts, societal needs and political interventions becomes more transparent so that interactive collaboration between them will be facilitated.

The formation and survival probability of negative carbon ions sputtered from a graphite sample was determined. The energy distribution of Cy ions was measured experimentally and the energy distribution of all sputtered carbon atoms was obtained from a computer simulation with the program TRIM.SP. From the two distributions, we obtained the probability that a carbon atom would capture an electron from the graphite and subsequently survive as it moves away from the surface. Ż. The results show two different situations. At low energy below 25 eV , the sputtered particles are all negatively ionized when the affinity level crosses the graphite Fermi level and afterwards, the population decay follows a semi-classical rate equation. Ż.At higher energy above 25 eV , a velocity-dependent fraction reaches the crossing distance as neutral atoms

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We describe our experimentation with the design and implementation of specific environments, consisting of heterogeneous computational, visualization, and control components. We illustrate the approach with the design of a problem–solving environment supporting the execution of genetic algorithms. We describe a prototype supporting parallel execution, visualization, and steering. A life cycle for the development of applications based on genetic algorithms is proposed.

Presacral vertebrae of a new Diplodocidae from the Late Jurassic Amoreira-Porto Novo Formation of Lourinhã, Portugal are described and figured. Dinheirosaurus lourinhanensis gen. et sp. is diagnosed by well developed accessory articulations derived from the hyposphene, exposed in lateral view; and by the smaller dorsoventral length of the lower section of the neural arch. It is considered that the organization of the dorsal neural arch of Dinheirosaurus is more derived than in Diplodocus, except in the dorsoventral development of the lower portion of it, which is higher (more derived) in the latter. Possibly the isolated geography of Portugal in the Late Jurassic gave rise to the distinct characters of this new genus.

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Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfide-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data. (C) 1999 Academic Press.

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