n/a

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F-peroxo, formed within 25 ms, which then leads to the release of H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, and O-2 and allowing the reaction to proceed for 70 ms when F-peroxo has reached its maximum accumulation, followed by spraying the reaction mixture into the H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F-peroxo. This observed parallel formation of multiple products (H2O2, diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F-peroxo) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.

Since the UN World Conference on Women in 1995 the discussion on gender has been extremely controverse. This issue of the Journal on Technology Assessment (from ITAS) presents and discuss the topic of gender research, and the relation between work and life and the changes that are occuring in the spheres of working lifes and daily lifes. Noch im Umfeld der 4. UN-Weltfrauenkonferenz in Peking 1995 wurde der Begriff gender extrem kontrovers diskutiert. Die Zeitschrift für Technikfolgenabschätzung des ITAS greift mit diesem Schwerpunkt erstmals das Thema „Genderforschung“ auf. Die Verknüpfung der beiden Themengebiete geht auf das Projekt: Wandel der Lebens- und Arbeitsbedingungen im Multimediabereich aus der Genderperspektive zurück, das zurzeit von ITAS im Auftrag des Sozialministerium Baden-Württemberg durchgeführt wird.

The resulting economic integration of industrial processes and manufacturing internationalisation lead several authors to argue that world economy is globalised. In this context, the approach to the divestment concept without an social and económical context, does not show a group of associated practices and representations. Choices and options are motivated by exogenous forces that pushes companies to determine strategies that stop capital investment on new equipment goods, or on other imaterial goods. This type of strategy is designated by "divestment". The social level of consequencies are not due to the closing down or de-localization of production units that are divesting, but can be materialised of efects that are irreversible. This means unemployment, de-skilling, labour precarization and even emergence of new forms of social exclusion in former industrialised regions.

Working teams in enterprise environment are considered as the most advanced forms of work organisation. This means the forms that can improve productivity quality of working life. Nevertheless, it prevail a slow development and dissemination of these advanced organisational forms in European companies. The reason for that lays in a complex linkage factors from social values to the economical pressures. But other factors are also related to the national systems of education training, to the different systems of industrial relations and technology policy.

Working teams in enterprise environment are considered as the most advanced forms of work organisation. This means the forms that can improve productivity quality of working life. Nevertheless, it prevail a slow development and dissemination of these advanced organisational forms in European companies. The reason for that lays in a complex linkage factors from social values to the economical pressures. But other factors are also related to the national systems of education training, to the different systems of industrial relations and technology policy.

The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes. It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form. The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions. The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4. It includes alternate conformations for 17 amino acid residues. At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified. With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations. Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible. For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin. At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.

Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe-4S] cluster per monomer. EPR and (1)H NMR data of recombinant and wild-type protein are compared.

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S](H)) and a binuclear Fe cluster ([2Fe](H)), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2), indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mossbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the CO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters, and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S]-[2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S](H) cluster from the 2+ to the 1+ state, followed by transfer of the reducing equivalent from the [4Fe-4S](H) subcluster to the binuclear [2Fe](H) subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S](H) and the [2Fe](H) subclusters. Implication of such a CO binding effect is discussed.

Unconjugated bilirubin is a neurotoxic pigment that interacts with membrane lipids. In this study we used electron paramagnetic resonance and the spin labels 5-, 7-, 12-, and 16-doxyl-stearic acid (DSA) to evaluate the depth of the hydrocarbon chain at which interaction of bilirubin preferentially occurs. In addition, we used different pH values to determine the molecular species involved. Resealed right-side-out ghosts were incubated (1-60 min) with bilirubin (3.4-42.8 microM) at pH 7.0, 7.4, and 8.0. Alterations of membrane dynamic properties were maximum after 15 min of incubation with 8.6 microM bilirubin at pH 7.4 and were accompanied by a significant release of phospholipids. Interestingly, concentrations of bilirubin up to 42.8 microM and longer incubations resulted in the elution of cholesterol and further increased that of phospholipids while inducing less structural alterations. Variation of the pH values from 8.0 to 7.4 and 7.0, under conditions of maximum perturbation, led to a change from an increased to a diminished polarity sensed by 5-DSA. Conversely, a progressive enhancement in fluidity was reported by 7-DSA, followed by 12- and 16-DSA. These results indicate that bilirubin while enhancing membrane lipid order at C-5 simultaneously has disordering effects at C-7. Furthermore, recovery of membrane dynamics after 15 min of bilirubin exposure along with the release of lipids is compatible with a membrane adaptive response to the insult. In addition, our data provide evidence that uncharged diacid is the species primarily interacting with the membrane as perturbation is favored by acidosis, a condition frequently associated with hyperbilirubinemia in premature and severely ill infants.

At the last National Conference of Industrial Sociology in Portugal (in March 1999) was presented a paper on the topic of “foresight as a technology and employment policy instrument” (A.B. Moniz) where there was a reference to the need of development of this kind of tools and instruments. This need is clear when one should neutralize the negative effects of such policies, and could support the positive influences, especially with the main aims of improvement of quality of working life. Thus, is today possible to make a first balance of the application in Portugal of a type of foresight analysis exercise: the Delphi method. After some of the uses of this method in Portugal on the fisheries socio-economical system and on the relation between information society and employment, we present in this paper a first assessment of such experiments and present some recommendations for future uses of this foresight technique. We conclude that they must be applied within the context of socio-economical perspectives, and not only the technological ones, although they should include that dimension.

Taking into account the global trends towards vertical de-integration and functional integration, the WorTiS project is expected to be able to determine to what extent the Portuguese automobile industry is experiencing far-reaching changes as far as innovative (post-fordist) work systems are concerned. One of the objectives is to reach wider conclusions regarding the sector under analysis within a new multi-disciplinary approach, in connection with other research networks (namely, GERPISA and IMVP-MIT). It will recover relevant information in automobile companies located in Portugal (like, Toyota, Citröen, FIAT, Renault, Ford, VW, UMM), in order to understand how concrete practices have being developed in time, and update the scientific knowledge with the development of new case studies (Mitsubishi, AutoEuropa, Opel-GM, and other sub-contracting firms). is intended to present a new fieldwork methodology in order to analyse the variety, and contradictory character, of changes in work practices. The analysis would focus on the cases of automotive firms that operated, and are still operating, in Portugal. The effectiveness of such a tool will last far beyond the project itself.

At the last National Conference of Industrial Sociology in Portugal (in March 1999) was presented a paper on the topic of “foresight as a technology and employment policy instrument” (A.B. Moniz) where there was a reference to the need of development of this kind of tools and instruments. This need is clear when one should neutralize the negative effects of such policies, and could support the positive influences, especially with the main aims of improvement of quality of working life. Thus, is today possible to make a first balance of the application in Portugal of a type of foresight analysis exercise: the Delphi method. After some of the uses of this method in Portugal on the fisheries socio-economical system and on the relation between information society and employment, we present in this paper a first assessment of such experiments and present some recommendations for future uses of this foresight technique. We conclude that they must be applied within the context of socio-economical perspectives, and not only the technological ones, although they should include that dimension.