Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.

The application of an external electric field to dry films of Asp-85-->Asn mutant bacteriorhodopsin causes deprotonation of the Schiff base, resulting in a shift of the optical absorption maximum from 600 nm to 400 nm. This is in marked contrast to the case of wild-type bacteriorhodopsin films, in which electric fields produce a red-shifted product whose optical properties are similar to those of the acid-blue form of the protein. This difference is due to the much weaker binding of the Schiff-base proton in the mutant protein, as indicated by its low pK of approximately 9, as compared with the value pK approximately 13 in the wild type. Other bacteriorhodopsins with lowered Schiff-base pK values should also exhibit a field-induced shift in the protonation equilibrium of the Schiff base. We propose mechanisms to account for these observations.

Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Precursores funcionalizantes de poros à base de alginato para obtenção de cerâmicas bioativas

A complexidade de desenvolver novas tecnologias para aplicações em reconstituição óssea se deve à necessidade de combinar várias propriedades químicas e físicas para que o material proporcione o desempenho almejado. Particularmente, em aplicações que visam osteogênese, os enxertos sintéticos devem ser bioativos, possuir porosidade com volume, geometria e interconectividade de poros controlados, além de ter boas propriedades mecânicas, dentro de limites relativamente rígidos. Por essa razão, o recobrimento de materiais bioinertes com cerâmicas bioativas se tornou o foco da presente pesquisa. O objetivo desse estudo foi desenvolver um novo método de produção de enxertos cerâmicos com macroporosidade funcionalizada, onde a formação e o revestimento dos poros são realizados em uma única etapa. Foi realizado o estudo de recobrimento com vidro bioativo e fosfato de cálcio. Para isso, agentes porogênicos na forma de grânulos (de 600 μm a 2 mm de diâmetro) foram sintetizados pelo método da gelificação de uma solução aquosa de alginato de sódio gotejada em solução de nitrato de cálcio (0,5 M), com incorporação de outros elementos para a formação de biovidro ou fosfato de cálcio. Esses grânulos foram conglomerados a um vidro ou alumina em pó, formando um compósito, que foi tratado termicamente para sinterização e formação de poros. No caso da matriz vítrea, a sinterização ocorreu com cristalização simultânea e concorrente. As cerâmicas resultantes foram caracterizadas por microscopia óptica e eletrônica de varredura, sendo possível observar a formação de macroporos aproximadamente esféricos (de 600 μm a 2 mm de diâmetro) revestidos internamente por uma camada de material com possível composição bioativa.

Changing Attitudes And Expectations In A University Setting: A Case Study

First and second year students enrolled in a four-year movement education based university Physical Education program completed a questionnaire regarding their expectations on entering university. In addition, graduates of the program were interviewed, one year after graduation, with regard to their understanding of and attitude towards movement education and how these had developed relative to their overall degree program. Most students had no knowledge of movement education prior to entering the program and the selection of this particular program was simply coincidental with their desire to pursue physical education. Whereas the students did participate in an activity course and a theory course in Year 1, it was only when participating in a Year 2 movement course which combined theory and practice within the same course that students recognized the movement base of the content. The progress of the students through the program reflects distinct declarative and procedural stages in knowledge development followed by an ability to generalize that knowledge a conceptual stage. The real understanding of movement education came as the students were required to teach movement education to students, children, and other groups.

Reasons withheld and insufficient reasoning as due process violations: two cases before the ECHR

Two very different cases decided by the European Court of Human Rights illustrate how the non-availability of sufficient reasons, for pre-trial judicial decisions in one case, and for a decision in a civil and administrative matter in the other, can lead to due process violations in terms of Articles 5 or 6 of the Convention of Human Rights and Fundamental Freedoms.

Managerialism at the bottom of the pyramid: a case study in Brazil

Nas últimas décadas, a Base da Pirâmide tem sido cada vez mais debatida entre líderes ocidentais como a nova terra das oportunidades. Com o advento do neoliberalismo no campo do desenvolvimento na década de 1990, o papel da sociedade civil e, em particular, o de Organizações Não-Governamentais, passou a ser enfatizado como sendo central nas estratégias ocidentais no "Sul" do mundo. Os atores do desenvolvimento, no entanto, muitas vezes abordaram essas questões utilizando perspectivas tradicionais, que estavam geralmente fora de contexto. A tese foca na controvérsia em torno do uso de técnicas de gestão em cenários que diferem daqueles nos quais estas ferramentas têm sido desenvolvidas. Em particular, ela procura compreender em que medida o gerencialismo - a ideologia da gestão - está a influenciando as atividades de uma Organização Não-Governamental brasileira, a Galpão Aplauso. O estudo, usando uma estrutura teórica, analisa o relacionamento entre a ONG e seus parceiros, sublinhando os resultados de conflitos ideológicos. No geral, descobriu-se como o encontro das perspectivas do Norte e do Sul originou alguns debates que levaram, em parte, à aceitação de ideias gerencialistas, tais como a replicabilidade e sistematização de processos, enquanto que em alguns casos, eles intensificaram a resistência da ONG sobre conceitos como sustentabilidade financeira e transformação em um negócio social.

The development and introduction of the automotive turbocharger: a case of innovation in response to fuel economy regulation. Final report.

National Highway Traffic Safety Administration, Office of Research and Development, Washington, D.C.

Capturing expert knowledge for threatened species assessments: a case study using NatureServe conservation status ranks

Assessments for assigning the conservation status of threatened species that are based purely on subjective judgements become problematic because assessments can be influenced by hidden assumptions, personal biases and perceptions of risks, making the assessment process difficult to repeat. This can result in inconsistent assessments and misclassifications, which can lead to a lack of confidence in species assessments. It is almost impossible to Understand an expert's logic or visualise the underlying reasoning behind the many hidden assumptions used throughout the assessment process. In this paper, we formalise the decision making process of experts, by capturing their logical ordering of information, their assumptions and reasoning, and transferring them into a set of decisions rules. We illustrate this through the process used to evaluate the conservation status of species under the NatureServe system (Master, 1991). NatureServe status assessments have been used for over two decades to set conservation priorities for threatened species throughout North America. We develop a conditional point-scoring method, to reflect the current subjective process. In two test comparisons, 77% of species' assessments using the explicit NatureServe method matched the qualitative assessments done subjectively by NatureServe staff. Of those that differed, no rank varied by more than one rank level under the two methods. In general, the explicit NatureServe method tended to be more precautionary than the subjective assessments. The rank differences that emerged from the comparisons may be due, at least in part, to the flexibility of the qualitative system, which allows different factors to be weighted on a species-by-species basis according to expert judgement. The method outlined in this study is the first documented attempt to explicitly define a transparent process for weighting and combining factors under the NatureServe system. The process of eliciting expert knowledge identifies how information is combined and highlights any inconsistent logic that may not be obvious in Subjective decisions. The method provides a repeatable, transparent, and explicit benchmark for feedback, further development, and improvement. (C) 2004 Elsevier SAS. All rights reserved.

Ethics and intervention: The 'humanitarian exception' and the problem of abuse in the case of Iraq

This article investigates the ethics of intervention and explores the decision to invade Iraq. It begins by arguing that while positive international law provides an important framework for understanding and debating the legitimacy of war, it does not cover the full spectrum of moral reasoning on issues of war and peace. To that end, after briefly discussing the two primary legal justifications for war (implied UN authorization and pre-emptive self-defence), and finding them wanting, it asks whether there is a moral 'humanitarian exceptions to this rule grounded in the 'just war' tradition. The article argues that two aspects of the broad tradition could be used to make a humanitarian case for war: the 'holy war' tradition and classical just war thinking based on natural law. The former it finds problematic, while the latter it argues provides a moral space to justify the use of force to halt gross breaches of natural law. Although such an approach may provide a moral justification for war, it also opens the door to abuse. It was this very problem that legal positivism from Vattel onwards was designed to address. As a result, the article argues that natural law and legal positivist arguments should be understood as complementary sets of ideas whose sometimes competing claims must be balanced in relation to particular cases. Therefore, although natural law may open a space for justifying the invasion of Iraq on humanitarian terms, legal positivism strictly limits that right. Ignoring this latter fact, as happened in the Iraq case, opens the door to abuse.

Dapsone-induced agranulocytosis. The role of xenobiotic-metabolizing enzymes demonstrated by a case report [Dapson-induzierte agranulozytose. Die rolle fremdstoffmetabolisierender enzyme am beispiel einer kasuistik]

A 34-year-old female patient with a three year history of generalized granuloma annulare was treated systemically with dapsone (DADPS). Six weeks after the onset of treatment, the patient developed an extensive tonsillitis of the base of the tongue with fever and malaise. Routine laboratory work showed a leukocytopenia with agranulocytosis. Further investigation revealed a marked decrease of the enzyme activity of N-acetyltransferase 2, which plays an important role in dapsone metabolism. Treatment included the cessation of dapsone, antibiotic coverage, and G-CSF leading to the rapid improvement of symptoms and normalization of leukocyte counts. Dapsone-induced angina agranulocytotica is a rare event and is interpreted as an idiosyncratic reaction. Depending on genetic polymorphisms of various enzymes, dapsone can be metabolized to immunologically or toxicologically relevant intermediates. Because of the risk of severe hematologic reactions, dapsone should only be employed for solid indications and with appropriate monitoring.

Using the Amazon metric to construct an image database based on what people do, not what they say

Current image database metadata schemas require users to adopt a specific text-based vocabulary. Text-based metadata is good for searching but not for browsing. Existing image-based search facilities, on the other hand, are highly specialised and so suffer similar problems. Wexelblat's semantic dimensional spatial visualisation schemas go some way towards addressing this problem by making both searching and browsing more accessible to the user in a single interface. But the question of how and what initial metadata to enter a database remains. Different people see different things in an image and will organise a collection in equally diverse ways. However, we can find some similarity across groups of users regardless of their reasoning. For example, a search on Amazon.com returns other products also, based on an averaging of how users navigate the database. In this paper, we report on applying this concept to a set of images for which we have visualised them using traditional methods and the Amazon.com method. We report on the findings of this comparative investigation in a case study setting involving a group of randomly selected participants. We conclude with the recommendation that in combination, the traditional and averaging methods would provide an enhancement to current database visualisation, searching, and browsing facilities.

Reasoning about timeouts

In real-time programming a timeout mechanism allows exceptional behaviour, such as a lack of response, to be handled effectively, while not overly affecting the programming for the normal case. For. example, in a pump controller if the water level has gone below the minimum level and the pump is on and hence pumping in more water, then the water level should rise above the minimum level within a specified time. If not, there is a fault in the system and it should be shut down and an alarm raised. Such a situation can be handled by normal case code that determines when the level has risen above the minimum, plus a timeout case handling the situation when the specified time to reach the minimum has passed. In this paper we introduce a timeout mechanism, give it a formal definition in terms of more basic real-time commands, develop a refinement law for introducing a timeout clause to implement a specification, and give an example of using the law to introduce a timeout. The framework used is a machine-independent real-time programming language, which makes use of a deadline command to represent timing constraints in a machine-independent fashion. This allows a more abstract approach to handling timeouts.

A case study on services upgrading prediction

Customer Base Analysis is perhaps the first stage of analysis in customer value, aiming to predict purchase frequency and customer lifecycle. An important part of the customer purchase frequency and its retention has to do with the service upgrade. Many models have tried to predict purchase frequency as well as upgrading. The comparison of these models seems important to provide academics with a picture of the current situation. The purpose of this research is to evaluate how models can predict service upgrade among a customer database of an online DVD rental company and suggest an alternative based on data mining techniques and data on historical transactions.

Techonology usage in the supply chain:the case of small 3PLs

In today's supply chain management practices, a successful strategy depends increasingly on the performance of third party logistics (3PLs) providers as they play a key integrative role linking the different supply chain elements more effectively. Information and communications technology (ICT) has become an important element of 3PL competitive capability as it enables higher levels of supply chain integration. Recent industry developments have widened the technological gap between large and small 3PLs. This is critical particularly for those markets populated by a large number of small 3PLs such as the Italian logistics industry. The purpose of this paper is to provide an overview of ICT adoption in small logistics service providers. The focus of the study is the Italian 3PL market. The methodology adopted in this paper is based on the action research framework and it is a combination of theoretical analysis and empirical findings (focus groups and surveys). Firstly, a literature review on ICT dissemination in the international and Italian 3PL industry has been carried out. Then, a field survey has been developed preceded by two focus groups. The empirical investigation examined a sample of 153 small Italian 3PLs on the base of a definition and taxonomy proposed by the authors. The results provide a useful technological profile of the surveyed companies, as well as an analysis of the role of ICT in customising services and of the factors influencing ICT adoption. To give a more robust scientific justification of the survey results, it would be useful to carry out an additional large-scale analysis including other countries with a high presence of small logistics service companies. Furthermore, a complementary case-study analysis would be of help in defining the role of specific factors in different types of 3PL. Further investigations in this field are needed to better understand innovation paths, and how ICT can improve the competitive capabilities of 3PLs and of the wider supply chain. Managers of small 3PLs can use the findings to set-up guidelines for improving a company's technology innovation. Technology vendors can use the findings to better market their service/product into the small logistics service provider segment. Despite the fact that much has been written about the dissemination of ICT, there is still a shortage of research in the field of small 3PLs with little empirical investigation into the usage of ICT by small 3PLs. This paper provides a contribution to filling this void and suggests some possible research directions.