Spectroscopic methods combined with density functional calculations are used to develop a detailed bonding description of the mu(4)-sulfide bridged tetranuclear Cu(Z) cluster in N(2)O reductase. The ground state of Cu(Z) has the 1Cu(II)/3Cu(I) configuration. The single electron hole dominantly resides on one Cu atom (Cu(I)) and partially delocalizes onto a second Cu atom (Cu(II)) via a Cu(I)-S-Cu(II) sigma/sigma superexchange pathway which is manifested by a Cu(II) --> Cu(I) intervalence transfer transition in absorption. The observed excited-state spectral features of Cu(Z) are dominated by the S --> Cu(I) charge-transfer transitions and Cu(I) based d-d transitions. The intensity pattern of individual S --> Cu(I) charge-transfer transitions reflects different bonding interactions of the sulfur valence orbitals with the four Cu's in the Cu(Z) cluster, which are consistent with the individual Cu-S force constants obtained from a normal coordinate analysis of the Cu(Z) resonance Raman frequencies and profiles. The Cu(I) d orbital splitting pattern correlates with its distorted T-shaped ligand field geometry and accounts for the observed low g( parallel ) value of Cu(Z) in EPR. The dominantly localized electronic structure description of the Cu(Z) site results from interactions of Cu(II) with the two additional Cu's of the cluster (Cu(III)/Cu(IV)), where the Cu-Cu electrostatic interactions lead to hole localization with no metal-metal bonding. The substrate binding edge of Cu(Z) has a dominantly oxidized Cu(I) and a dominantly reduced Cu(IV). The electronic structure description of Cu(Z) provides a strategy to overcome the reaction barrier of N(2)O reduction at this Cu(I)/Cu(IV) edge by simultaneous two-electron transfer to N(2)O in a bridged binding mode. One electron can be donated directly from Cu(IV) and the other from Cu(II) through the Cu(II)-S-Cu(I) sigma/sigma superexchange pathway. A frontier orbital scheme provides molecular insight into the catalytic mechanism of N(2)O reduction by the Cu(Z) cluster.

The enzyme hydrogenase isolated from the sulphate reducing anaerobic bacterium Desulfovibrio gigas was encapsulated in reverse micelles of AOT-water-isooctane. The enzyme ability to consume molecular hydrogen was studied as a function of the micelle size (given by W-o = [H2O]/[organic solvent]). A peak of catalytic activity was obtained for W-o = 18, a micelle size theoretically fitting the heterodimeric hydrogenase molecule. At this W-o value, the recorded catalytic activity was slightly higher than in a buffer system (K-cat = 169.43 s(-1) against the buffer value of 151 s(-1)). The optimal buffer used to encapsulate the enzyme was found to be imidazole 50 mM, pH 9.0, The molecular hydrogen production activity was also tested in this reverse micelle medium. (C) 2002 Elsevier Science Inc. All lights reserved.

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

Using a myosin preparation containing endogenous myosin light-chain (LC2) kinase and phosphatase and calmodulin, i.e. near physiological ones, the interaction of vanadate oligomers with phosphorylated myosin was evaluated. Decavanadate or metavanadate solutions (2-15 mM total vanadate) did not prevent the phosphorylation state of the regulatory myosin lightchain, as observed by urea-polyacrylamide gel electrophoresis. The relative order of line broadening upon protein addition, reflecting the interaction of the vanadate oligomers with phosphorylated myosin, was V10 > V-4 > V-1 = 1 whereas, no changes were observed for monomeric vanadate. In the presence of ATP, V-1 signal was shifted upfield 2 ppm and became broadened, while V4 signal became narrowed. Moreover, a significant increase in myosin ATPase inhibition (60%) was observed when decameric vanadate species were present (1.4 mM). It is concluded that, under conditions near physiological ones, decameric vanadate differs from vanadate oligomers present in metavanadate solutions due to its strong interaction with the phosphorylated enzyme and myosin ATPase inhibition. Besides, ATP decreases the affinity of myosin for tetravanadate, induces the interaction with monomeric vanadate, whereas it does not affect decameric vanadate interaction. (C) 2002 Elsevier Science B.V. All rights reserved.

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.

This article is based on the lesson presented in the scope of academy activities in the area of Sociology (at FCT-UNL). It is intended to approach the controversies concerning the relation between technology and society (technological determinism, effect on employment, importance of the social behaviours in the definition of needs for new products and equipment), and on the most recent trends (over all, since middle of the last century) in terms of technological evolution and of its social and cultural change. Finally, this subject was dedicated to the presentation of the main factors that has lead to the development of the contribution of sociology for the training and education in engineering. Thus, one intends to acquire new elements on this area of knowledge also presented in other schools of engineering of other countries (for examples, United States and Holland), and how the theoretical beddings are been developed since the beginning of 20th century on the modalities of work organization that imply a cooperation between engineering and social sciences.

This article is based on the lesson presented in the scope of academy activities in the area of Sociology (at FCT-UNL). It is intended to approach the controversies concerning the relation between technology and society (technological determinism, effect on employment, importance of the social behaviours in the definition of needs for new products and equipment), and on the most recent trends (over all, since middle of the last century) in terms of technological evolution and of its social and cultural change. Finally, this subject was dedicated to the presentation of the main factors that has lead to the development of the contribution of sociology for the training and education in engineering. Thus, one intends to acquire new elements on this area of knowledge also presented in other schools of engineering of other countries (for examples, United States and Holland), and how the theoretical beddings are been developed since the beginning of 20th century on the modalities of work organization that imply a cooperation between engineering and social sciences.

BACKGROUND/AIMS: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. METHODS: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. RESULTS: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P<0.01), as well as disrupted protein mobility (P<0.001). Consistent with increased permeability, cytochrome c was released from the intermembrane space (P<0.01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P<0.01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. CONCLUSIONS: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

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Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe2+ chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.

Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.

Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.

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Recent studies continue to indicate the existence of a narrow relationship between flexible work organizations and the economic growth, in particular, Sweden and Germany. The measure of this relationship is many times the result of the value added per worker. Therefore, the causes of economic growth must be perceived from the interior of the company (work organization, technology, infrastructures, product design). On the other hand, the capacity of innovation can be perceived by the market through new products and services, and still significant changes with the introduction of new equipment and design of an efficient work organization. In this article it is analyzed the evolution of the productivity and employment levels in Portugal and other European countries, over all, during the decade of 90. An analysis for sector is made still. One verifies often that economic growth can be without employment growth. However, the growth can be obtained when if it reaches bigger added value and the efficiency can be verified when there are diminished costs for a same period of time, although that the labor productivity is also the pointer of the quality of life in a economy, therefore it represents the value produced by the work. Moreover, the total factors productivity is the measure of the technological and organizational progress (don’t includes only the technological investments). It is tried to get and to analyze the available statistics on these dimension in Portugal, concluding that Portugal presents an enterprise structure predominantly based on small and very small companies, a great number of which is market by a weak potential of adaptability, innovation and sustainability. It becomes urgent to take the non-material factors that integrate the productivity dimension, as factors of intervention for a benchmarking that allows a process of sustainable growth.

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.

The Portuguese National Action Plan for Employment (NAP) is the main instrument for the labour market policy. It was adopted in 1998. The NAP transposes to the Portuguese reality the contents of such guidelines, with the adequate adjustments required by the national specificities, establishing objectives, quantified targets and action deadlines, as well as defining new programmes and measures. Therefore, the objectives of the labour market policy were focusing on the promotion of an adequate transition of the youngsters in active life, on the promotion of social and professional insertion and fight against long term unemployment and exclusion, on the improvement of basic and professional qualification of the working population in a perspective of lifelong training, namely as a way to prevent unemployment phenomena, and on the preventive management and follow-up of sectoral restructuring processes.

Recent studies continue to indicate the existence of a narrow relationship between flexible work organizations and the economic growth, in particular, Sweden and Germany. The measure of this relationship is many times the result of the value added per worker. Therefore, the causes of economic growth must be perceived from the interior of the company (work organization, technology, infrastructures, product design). On the other hand, the capacity of innovation can be perceived by the market through new products and services, and still significant changes with the introduction of new equipment and design of an efficient work organization. In this article it is analyzed the evolution of the productivity and employment levels in Portugal and other European countries, over all, during the decade of 90. An analysis for sector is made still. One verifies often that economic growth can be without employment growth. However, the growth can be obtained when if it reaches bigger added value and the efficiency can be verified when there are diminished costs for a same period of time, although that the labor productivity is also the pointer of the quality of life in a economy, therefore it represents the value produced by the work. Moreover, the total factors productivity is the measure of the technological and organizational progress (don’t includes only the technological investments). It is tried to get and to analyze the available statistics on these dimension in Portugal, concluding that Portugal presents an enterprise structure predominantly based on small and very small companies, a great number of which is market by a weak potential of adaptability, innovation and sustainability. It becomes urgent to take the non-material factors that integrate the productivity dimension, as factors of intervention for a benchmarking that allows a process of sustainable growth.

The Portuguese National Action Plan for Employment (NAP) is the main instrument for the labour market policy. It was adopted in 1998. The NAP transposes to the Portuguese reality the contents of such guidelines, with the adequate adjustments required by the national specificities, establishing objectives, quantified targets and action deadlines, as well as defining new programmes and measures. Therefore, the objectives of the labour market policy were focusing on the promotion of an adequate transition of the youngsters in active life, on the promotion of social and professional insertion and fight against long term unemployment and exclusion, on the improvement of basic and professional qualification of the working population in a perspective of lifelong training, namely as a way to prevent unemployment phenomena, and on the preventive management and follow-up of sectoral restructuring processes.

A propose for this article is to present information on the emergence of the knowledge field of sociology of work and labor studies in Portugal. The period of 1974-1984 produced an interesting bibliography in the fields of social stratification, labor conflicts and social history. However, and since the mid-1980s, significant changes have emerged. There took place an increased theoretical diversity and also there was a major preoccupation with the development of a more professional teaching of the discipline. At this time, the first graduated specialists in Sociology appeared in the labor market since 1980, working in municipalities, industrial firms, public health institutions, schools, or even in research and development (R&D) units. The Portuguese association in this field (APSIOT) organized many scientific meetings, debates with unionists, managers and politicians, beside the regular publication of review “Organiza{\c c}ões e Trabalho” (Organization and Work) since 1989. It started the diffusion of a professional culture of sociologists who associate science and their occupation concerning themselves not only with scientific quality, but also with technical competence and social responsibility. At the same time, one could assist to the participation of sociologists in the social and organizational molding of new technologies in order to promote alternative production systems.

A propose for this article is to present information on the emergence of the knowledge field of sociology of work and labor studies in Portugal. The period of 1974-1984 produced an interesting bibliography in the fields of social stratification, labor conflicts and social history. However, and since the mid-1980s, significant changes have emerged. There took place an increased theoretical diversity and also there was a major preoccupation with the development of a more professional teaching of the discipline. At this time, the first graduated specialists in Sociology appeared in the labor market since 1980, working in municipalities, industrial firms, public health institutions, schools, or even in research and development (R&D) units. The Portuguese association in this field (APSIOT) organized many scientific meetings, debates with unionists, managers and politicians, beside the regular publication of review “Organizações e Trabalho” (Organization and Work) since 1989. It started the diffusion of a professional culture of sociologists who associate science and their occupation concerning themselves not only with scientific quality, but also with technical competence and social responsibility. At the same time, one could assist to the participation of sociologists in the social and organizational molding of new technologies in order to promote alternative production systems.