Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [H-3] palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [H-3] palmitate and metabolites were measured in four experimentalgroups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [H-3] palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [H-3] palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Intramolecular electron transfer in a bacterial sulfite dehydrogenase

Sulfite dehydrogenase (SDH) from Starkeya novella, a sulfite-oxidizing molybdenum-containing enzyme, has a novel tightly bound αβ-heterodimeric structure in which the Mo cofactor and the c-type heme are located on different subunits. Flash photolysis studies of intramolecular electron transfer (IET) in SDH show that the process is first-order, independent of solution viscosity, and not inhibited by sulfate, which strongly indicates that IET in SDH proceeds directly through the protein medium and does not involve substantial movement of the two subunits relative to each other. The IET results for SDH contrast with those for chicken and human sulfite oxidase (SO) in which the molybdenum domain is linked to a b-type heme domain through a flexible loop, and IET shows a remarkable dependence on sulfate concentration and viscosity that has been ascribed to interdomain docking. The results for SDH provide additional support for the interdomain docking hypothesis in animal SO and clearly demonstrate that dependence of IET on viscosity and sulfate is not an inherent property of all sulfite-oxidizing molybdenum enzymes.

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Model studies on the role of citrate, malate and pectin esterification on the enzymatic degradation of Al- and Ca-pectate gels: possible implications for Al-tolerance

Aluminium (At) tolerance in plants may be conferred by reduced binding of Al in the cell wall through low root cation exchange capacity (CEC) or by organic acid exudation. Root CEC is related to the degree of esterification (DE) of pectin in the cell wall, and pectin hydrolysis plays a role in cell expansion. Therefore, it was hypothesised that Al-tolerant plants with a low root CEC maintain pectin hydrolysis in the presence of Al, allowing cell expansion to continue. Irrespective of the DE, binding of Al to pectin reduced the enzymatic hydrolysis of Al-pectin gels by polygalacturonase (E.C. 3.2.1.15). Pectin gels with calcium (Ca) were slightly hydrolysed by polygalacturonase. It was concluded, therefore, that Al tolerance conferred by low root CEC is not mediated by the ability to maintain pectin hydrolysis. Citrate and malate, but not acetate, effectively dissolved Al-pectate gel and led to hydrolysis of the dissolved pectin by polygalacturonase. The organic acids did not dissolve Ca-pectate, nor did they increase pectin hydrolysis by polygalacturonase. It was concluded that exudation of some organic acids can remove Al bound to pectin and this could alleviate toxicity, constituting a tolerance mechanism. (C) 2003 Editions scientitiques et medicales Elsevier SAS. All rights reserved.

A review of drainage and spontaneous rupture in free standing thin films with tangentially immobile interfaces

A review of spontaneous rupture in thin films with tangentially immobile interfaces is presented that emphasizes the theoretical developments of film drainage and corrugation growth through the linearization of lubrication theory in a cylindrical geometry. Spontaneous rupture occurs when corrugations from adjacent interfaces become unstable and grow to a critical thickness. A corrugated interface is composed of a number of waveforms and each waveform becomes unstable at a unique transition thickness. The onset of instability occurs at the maximum transition thickness, and it is shown that only upper and lower bounds of this thickness can be predicted from linear stability analysis. The upper bound is equivalent to the Freakel criterion and is obtained from the zeroth order approximation of the H-3 term in the evolution equation. This criterion is determined solely by the film radius, interfacial tension and Hamaker constant. The lower bound is obtained from the first order approximation of the H-3 term in the evolution equation and is dependent on the film thinning velocity A semi-empirical equation, referred to as the MTR equation, is obtained by combining the drainage theory of Manev et al. [J. Dispersion Sci. Technol., 18 (1997) 769] and the experimental measurements of Radoev et al. [J. Colloid Interface Sci. 95 (1983) 254] and is shown to provide accurate predictions of film thinning velocity near the critical thickness of rupture. The MTR equation permits the prediction of the lower bound of the maximum transition thickness based entirely on film radius, Plateau border radius, interfacial tension, temperature and Hamaker constant. The MTR equation extrapolates to Reynolds equation under conditions when the Plateau border pressure is small, which provides a lower bound for the maximum transition thickness that is equivalent to the criterion of Gumerman and Homsy [Chem. Eng. Commun. 2 (1975) 27]. The relative accuracy of either bound is thought to be dependent on the amplitude of the hydrodynamic corrugations, and a semiempirical correlation is also obtained that permits the amplitude to be calculated as a function of the upper and lower bound of the maximum transition thickness. The relationship between the evolving theoretical developments is demonstrated by three film thickness master curves, which reduce to simple analytical expressions under limiting conditions when the drainage pressure drop is controlled by either the Plateau border capillary pressure or the van der Waals disjoining pressure. The master curves simplify solution of the various theoretical predictions enormously over the entire range of the linear approximation. Finally, it is shown that when the Frenkel criterion is used to assess film stability, recent studies reach conclusions that are contrary to the relevance of spontaneous rupture as a cell-opening mechanism in foams. (C) 2003 Elsevier Science B.V. All rights reserved.

Continuous variable polarization entanglement, experiment and analysis

We generate and characterize continuous variable polarization entanglement between two optical beams. We first produce quadrature entanglement, and by performing local operations we transform it into a polarization basis. We extend two entanglement criteria, the inseparability criteria proposed by Duan et al (2000 Phys. Rev. Lett. 84 2722) and the Einstein–Podolsky–Rosen (EPR) paradox criteria proposed by Reid and Drummond (1988 Phys. Rev. Lett. 60 2731), to Stokes operators; and use them to characterize the entanglement. Our results for the EPR paradox criteria are visualized in terms of uncertainty balls on the Poincaré sphere. We demonstrate theoretically that using two quadrature entangled pairs it is possible to entangle three orthogonal Stokes operators between a pair of beams, although with a bound √3 times more stringent than for the quadrature entanglement.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Novas tend??ncias do mercado de trabalho

Na ruptura social fordista, o desemprego cresce bruscamente, fazendo surgir configura????es vari??veis, onde o conceito de press??o salarial passa a designar o complemento monet??rio indireto, destinado a assegurar a reprodu????o intergeneracional nas sociedades capitalistas plenamente desenvolvidas. A nova rela????o salarial compreende a rela????o de emprego (rela????o monet??ria direta) e a press??o salarial (rela????o monet??ria indireta). O aparecimento de rela????es de trabalho prec??rias for??a a press??o salarial a substituir a prote????o social em decad??ncia. Dos arranjos em forma????o, impor-se-??o os que encontrarem uma coer??ncia sist??mica m??nima. O poder p??blico agir?? como regulador da rela????o salarial e garantidor ??ltimo da press??o salarial, respeitando a flexibilidade do aparelho produtivo e a mobilidade e flexibilidade do trabalho. O autor tra??a trajet??rias nacionais baseados em 2 cen??rios estilizados, que s??o a variante americana (modelo t??pico da desregulamenta????o e flexibilidade externa) e a variante europ??ia (modelo t??pico da gest??o institucionalizada e flexibilidade interna).

A educação em cena : a Revista de Educação como circulação de representações sobre saberes educacionais no Espírito Santo (1934-1937)

Este estudo analisa os processos de circulação e apropriação das representações sobre os saberes da educação escolarizada difundidos pela Revista de Educação do Espírito Santo, entre os anos de 1934 e 1937. Como referencial teórico baseamo- nos em Chartier (1990) acerca do conceito de representação, em Balandier (1982) em relação ao conceito de encenação de poder instituído que assume visibilidade quando circula na Revista, concedendo publicização aos feitos políticos realizados por dado grupo social, e em Julia (2001), junto ao conceito de cultura escolar. A partir desse arcabouço teórico, empreendemos metodologia de pesquisa a partir da análise histórica da fonte, dialogando, para tanto, com diferentes documentos e registros que configuram uma série de dados que constituem nossa fonte. Trata-se da análise das representações travadas no debate sobre a formação, divulgação e apropriação do conjunto de práticas e saberes pedagógicos dirigido aos professores, por parte de um grupo de intelectuais locais que se apresentava como portador do projeto de modernização do Espírito Santo inserido no contexto nacional. A Revista de Educação/ES tinha entre seus principais objetivos o de (in)formar os professores, ou seja, enquadrar suas práticas às novas demandas educacionais. Deste modo, a Revista esteve atrelada a um projeto educacional em que os intelectuais corroboravam a ampla circulação de um conjunto de representações sobre a modernidade, utilizando a Revista como suporte, visto que, desde as capas, são expostos “monumentos de modernização”, como os prédios escolares e todo um complexo arranjo de artefatos simbólicos, traduzidos muitas vezes em festas e rituais escolares que evocavam “um novo tempo para a Educação do Estado”, ou seja, fazendo da educação escolarizada um espetáculo.

Sensor-driven agenda for intelligent home care of the elderly

The exponential increase of home-bound persons who live alone and are in need of continuous monitoring requires new solutions to current problems. Most of these cases present illnesses such as motor or psychological disabilities that deprive of a normal living. Common events such as forgetfulness or falls are quite common and have to be prevented or dealt with. This paper introduces a platform to guide and assist these persons (mostly elderly people) by providing multisensory monitoring and intelligent assistance. The platform operates at three levels. The lower level, denominated ‘‘Data acquisition and processing’’performs the usual tasks of a monitoring system, collecting and processing data from the sensors for the purpose of detecting and tracking humans. The aim is to identify their activities in an intermediate level called ‘‘activity detection’’. The upper level, ‘‘Scheduling and decision-making’’, consists of a scheduler which provides warnings, schedules events in an intelligent manner and serves as an interface to the rest of the platform. The idea is to use mobile and static sensors performing constant monitoring of the user and his/her environment, providing a safe environment and an immediate response to severe problems. A case study on elderly fall detection in a nursery home bedroom demonstrates the usefulness of the proposal.

O planejamento econômico no Brasil: considerações críticas

In the past thirty years, a series of plans have been developed by successive Brazilian governments in a continuing effort to maximize the nation's resources for economic and social growth. This planning history has been quantitatively rich but qualitatively poor. The disjunction has stimulated Professor Mello e Souza to address himself to the problem of national planning and to offer some criticisms of Brazilian planning experience. Though political instability has obviously been a factor promoting discontinuity, his criticisms are aimed at the attitudes and strategic concepts which have sought to link planning to national goals and administration. He criticizes the fascination with techniques and plans to the exclusion of proper diagnosis of the socio-political reality, developing instruments to coordinate and carry out objectives, and creating an administrative structure centralized enough to make national decisions and decentralized enough to perform on the basis of those decisions. Thus, fixed, quantified objectives abound while the problem of functioning mechanisms for the coordinated, rational use of resources has been left unattended. Although his interest and criticism are focused on the process and experience of national planning, he recognized variation in the level and results of Brazilian planning. National plans have failed due to faulty conception of the function of planning. Sectorial plans, save in the sector of the petroleum industry under government responsibility, ha e not succeeded in overcoming the problems of formulation and execution thereby repeating old technical errors. Planning for the private sector has a somewhat brighter history due to the use of Grupos Executivos which has enabled the planning process to transcend the formalism and tradition-bound attitudes of the regular bureaucracy. Regional planning offers two relatively successful experiences, Sudene and the strategy of the regionally oriented autarchy. Thus, planning history in Brazil is not entirely black but a certain shade of grey. The major part of the article, however, is devoted to a descriptive analysis of the national planning experience. The plans included in this analysis are: The Works and Equipment Plan (POE); The Health, Food, Transportation and Energy Plan (Salte); The Program of Goals; The Trienal Plan of Economic and Social Development; and the Plan of Governmental Economic Action (Paeg). Using these five plans for his historical experience the author sets out a series of errors of formulation and execution by which he analyzes that experience. With respect to formulation, he speaks of a lack of elaboration of programs and projects, of coordination among diverse goals, and of provision of qualified staff and techniques. He mentions the absence of the definition of resources necessary to the financing of the plan and the inadequate quantification of sectorial and national goals due to the lack of reliable statistical information. Finally, he notes the failure to coordinate the annual budget with the multi-year plans. He sees the problems of execution as beginning in the absence of coordination between the various sectors of the public administration, the failure to develop an operative system of decentralization, the absence of any system of financial and fiscal control over execution, the difficulties imposed by the system of public accounting, and the absence of an adequate program of allocation for the liberation of resources. He ends by pointing to the failure to develop and use an integrated system of political economic tools in a mode compatible with the objective of the plans. The body of the article analyzes national planning experience in Brazil using these lists of errors as rough model of criticism. Several conclusions emerge from this analysis with regard to planning in Brazil and in developing countries, in general. Plans have generally been of little avail in Brazil because of the lack of a continuous, bureaucratized (in the Weberian sense) planning organization set in an instrumentally suitable administrative structure and based on thorough diagnoses of socio-economic conditions and problems. Plans have become the justification for planning. Planning has come to be conceived as a rational method of orienting the process of decisions through the establishment of a precise and quantified relation between means and ends. But this conception has led to a planning history rimmed with frustration, and failure, because of its rigidity in the face of flexible and changing reality. Rather, he suggests a conception of planning which understands it "as a rational process of formulating decisions about the policy, economy, and society whose only demand is that of managing the instrumentarium in a harmonious and integrated form in order to reach explicit, but not quantified ends". He calls this "planning without plans": the establishment of broad-scale tendencies through diagnosis whose implementation is carried out through an adjustable, coherent instrumentarium of political-economic tools. Administration according to a plan of multiple, integrated goals is a sound procedure if the nation's administrative machinery contains the technical development needed to control the multiple variables linked to any situation of socio-economic change. Brazil does not possess this level of refinement and any strategy of planning relevant to its problems must recognize this. The reforms which have been attempted fail to make this recognition as is true of the conception of planning informing the Brazilian experience. Therefore, unworkable plans, ill-diagnosed with little or no supportive instrumentarium or flexibility have been Brazil's legacy. This legacy seems likely to continue until the conception of planning comes to live in the reality of Brazil.

Optical properties and transport in PLD-GaN

We present structural, optical and transport data on GaN samples grown by hybrid, two-step low temperature pulsed laser deposition. The band gap of samples with good crystallinity has been deduced from optical spectra. Large below gap band tails were observed. In samples with the lowest crystalline quality the PL spectra are quite dependent on spot laser incidence. The most intense PL lines can be attributed to excitons bounded to stacking faults. When the crystalline quality of the samples is increased the ubiquitous yellow emission band can be detected following a quenching process described by a similar activation energy to that one found in MOCVD grown samples. The samples with the highest quality present, besides the yellow band, show a large near band edge emission which peaked at 3.47 eV and could be observed up to room temperature. The large width of the NBE is attributed to effect of a wide distribution of band tail states on the excitons. Photoconductivity data supports this interpretation.