Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Diversity in the disulfide folding pathways of cystine knot peptides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides; poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.

Responsibility to protect or Trojan Horse? The crisis in darfur and humanitarian intervention after Iraq

What does the world's engagement with the unfolding crisis in Darfur tell us about the impact of the Iraq war on the norm of humanitarian intervention? Is a global consensus about a "responsibility to protect" more or less likely? There are at least three potential answers to these questions. Some argue that the merging of strategic interests and humanitarian goods amplified by the intervention in Afghanistan makes it more likely that the world's most powerful states will act to prevent or halt humanitarian crises. Others insist that the widespread perception that the United States and its allies "abused" humanitarian justifications to legitimate its invasion of Iraq has set back efforts to build a global consensus about humanitarian action. A third group argues that the "responsibility to protect" inhibits the potential for abuse and, as a result, consensus is likely to strengthen post-Iraq for precisely this reason. Through a detailed study of the international engagement with Darfur, I suggest that the latter two arguments have merit but need to be adjusted. I argue that the humanitarian intervention norm has changed in two subtle ways. First, while the strength of the norm itself has not changed, the credibility of the United States and U.K. as "norm carriers" has been significantly undermined. Second, while the "responsibility to protect" has been invoked to support international activism, it has also re-legitimated anti-interventionist arguments.

Hyperspace geography: Visualizing fitness landscapes beyond 4D

Human perception is finely tuned to extract structure about the 4D world of time and space as well as properties such as color and texture. Developing intuitions about spatial structure beyond 4D requires exploiting other perceptual and cognitive abilities. One of the most natural ways to explore complex spaces is for a user to actively navigate through them, using local explorations and global summaries to develop intuitions about structure, and then testing the developing ideas by further exploration. This article provides a brief overview of a technique for visualizing surfaces defined over moderate-dimensional binary spaces, by recursively unfolding them onto a 2D hypergraph. We briefly summarize the uses of a freely available Web-based visualization tool, Hyperspace Graph Paper (HSGP), for exploring fitness landscapes and search algorithms in evolutionary computation. HSGP provides a way for a user to actively explore a landscape, from simple tasks such as mapping the neighborhood structure of different points, to seeing global properties such as the size and distribution of basins of attraction or how different search algorithms interact with landscape structure. It has been most useful for exploring recursive and repetitive landscapes, and its strength is that it allows intuitions to be developed through active navigation by the user, and exploits the visual system's ability to detect pattern and texture. The technique is most effective when applied to continuous functions over Boolean variables using 4 to 16 dimensions.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Perceptions of organizational change: A stress and coping perspective

Few organizational change studies identify the aspects of change that are salient to individuals and that influence well-being. The authors identified three distinct change characteristics: the frequency, impact and planning of change. R. S. Lazarus and S. Folkman's (1984) cognitive phenomenological model of stress and coping was used to propose ways that these change characteristics influence individuals' appraisal of the uncertainty associated with change, and, ultimately, job satisfaction and turnover intentions. Results of a repeated cross-sectional study that collected individuals' perceptions of change one month prior to employee attitudes in consecutive years indicated that while the three change perceptions were moderately to strongly intercorrelated, the change perceptions displayed differential relationships with outcomes. Discussion focuses on the importance of systematically considering individuals' subjective experience of change.

A DIALÉTICA DO DESESPERO EM KIERKEGAARD E SUA INFLUÊNCIA SOBRE O CONCEITO DE ALIENAÇÃO EM PAUL TILLICH

A respectiva pesquisa procurou estabelecer a relação de influência da filosofia de Søren Kierkegaard sobre a construção teológica de Paul Tillich. Para tanto, utilizamos como pano de fundo ou estrutura argumentativa o conceito de Desespero humano de Kierkegaard, comparando-o ao conceito de Alienação do ser de Tillich. Objetivamos com isso, demonstrar o entrelaçamento dialogal composto no desenvolvimento da chamada doutrina do pecado em ambos os autores. Isto é, como o desdobramento da antropologia kierkegaardiana problematizou os fundamentos da teologia sistemática, sobretudo, na questão da harmatiologia, e, como esta mesma problematização, foi capaz de padronizar o modus operandi que determinou a elaboração feita por Tillich sobre o mesmo assunto. Obviamente, que, no decorrer dessa investigação, não se pretendeu desqualificar ou eliminar a originalidade dos autores, nem tampouco transformar seus argumentos em uma espécie de amálgama conceitual. Assim, antes de uma suposta crítica de ter se proposto uma fusão arbitrária de ideias, buscamos, mais uma vez apontar, soluções de dialéticas e centros comuns, que apontem para aqueles elementos de convergência, aonde de fato, o diálogo é possível.

A SOTERIOLOGIA SOCIAL DE JOHN WESLEY com consideração especial de seus aspectos comunitários, sinergéticos e públicos

Este estudo investiga os elementos da soteriologia de John Wesley e seus aspectos dinamizantes. Em vez de favorecer um determinado período da vida de Jonh Wesley, a biografia teológica de John Wesley é desenvolvida a partir do conceito da sedimentação de experiência. Dessa forma, a theologia viatorum de John Wesley é desenvolvida com foco nos seus elementos transversais e os crescentes desdobramentos dos mesmos segundo os seus aspectos comunitários, sinergéticos e públicos. Segundo esta tese, a theologia viatorum de John Wesley ganha em profundidade e vitalidade com a sua crescente e consciente aprendizagem e compromisso com o povo inglês e, em especial, com os pobres da Inglaterra. Acompanhado por uma cuidadosa auto-análise, Wesley parte para a análise do efeito de diversas soteriologias dos seus dias no cotidiano, as quais são marcadas pela demorada transição entre a época medieval e a modernidade. Surge uma re-construção característica da soteriologia, designado por nós como soteriologia social. Entendemos esta soteriologia social como o eixo principal da teologia de John Wesley que se manifesta numa típica antropologia, cristologia, pneumatologia, escatologia e eclesiologia soteriológica. Os aspectos comunitários, sinergéticos e públicos desta soteriologia social são vistos e desenvolvidos por Wesley a partir da auto-experiência e da convivência com o povo, suas dinâmicas, sua cultura, suas necessidades e suas competências. Tais aspectos levam, passo a passo, a uma práxis que começa a contemplar o entrelaçamento entre a reforma da igreja, da nação e a transformação de pessoas. Este movimento é comotivado por um horizonte soterioló gico utópico que prevê a continuação da theologia viatorum até na transformação do cosmo inteiro. A soteriologia social é parecida com a um tecido que acompanha a topografia socio-econômica e religiosa da época, procurando caminhos para a superação dos seus impasses, da sua desesperança e irresponsabilidade em relação aos pobres.(AU)

CORPO E COTIDIANO: A EXPERIÊNCIA DE MULHERES DE MOVIMENTOS POPULARES DESAFIA A TEOLOGIA FEMINISTA DA LIBERTAÇÃO NA AMÉRICA LATINA

A experiência das mulheres tem sido afirmada como ponto de partida de Teologias Feministas da Libertação. Na América Latina, o sexismo, a feminização da pobreza, a violência contra as mulheres têm inspirado o movimento feminista a se articular com as mulheres de movimentos populares para a conquista de direitos plenos para as mulheres. As Teologias da Libertação não lograram analisar a opressão das mulheres e apoiar sua luta, pois entenderam sua opressão como um desdobramento da exploração econômica dos países pobres pelos países ricos. Queremos demonstrar que a Teologia Feminista da Libertação, ao contrário, utilizando um método de análise feminista, é capaz de oferecer um suporte teológico às reivindicações das mulheres latino-americanas. O presente trabalho vai analisar teologicamente a experiência das mulheres de movimentos populares e explorar dois temas prioritários dessa experiência: corpo e cotidiano. Assim procedendo, pretende demonstrar que corpo e cotidiano são importantes categorias para a Teologia Feminista da Libertação na América Latina, através das quais ela poderá contribuir para apoiar a busca da autonomia e empoderamento das mulheres, em especial das mulheres pobres.(AU)

Do grande Outro ao Deus que falta Um estudo sobre a falta e as concepções de Deus e de religião no pensamento de Jacques Lacan

O tema desta tese é a concepção de Deus e de religião no pensamento de Jacques Lacan. Partindo de um estudo dos principais conceitos da psicanálise lacaniana, articulados em torno dos registros do imaginário, do simbólico e do real, a tese aborda as diferentes expressões da dimensão da falta que caracteriza a condição do ser humano e as articula com sua incidência sobre o desenvolvimento das concepções de Deus e de religião em Lacan. Constata que Lacan trabalha com duas concepções distintas de Deus: Uma relacionada com os registros do imaginário e do simbólico, onde Deus se caracteriza como aquele que se constitui pelo sacrifício do desejo do sujeito oferecido a ele como forma de negar a falta é o Deus que goza do sujeito. A outra está relacionada ao registro do real. É o Deus que se manifesta no sintoma, que porta um enigma e uma falta, suscitando o desejo no sujeito. Lacan o identifica como o Deus de Moisés. Essas diferentes concepções de Deus permitem uma crítica à concepção lacaniana de religião, bem como, a identificação de pontos de proximidade entre esta e a psicanálise.

A “REVOLTA” DE JEÚ E A ESTELA DE DÃ: Um Estudo Em 2 Reis 10,28-36

A pesquisa tem por objetivo trabalhar o evento da Revolta de Jeú, em conjunto com a Estela de Dã, tendo como ponto de partida para tal, a exegese da perícope de 2 Reis 10-28,36. A história Deuteronomista apresenta o ato da Revolta de Jeú como sendo um feito demasiadamente importante, na restauração do culto a Javé em Israel, a partir de um contexto onde o culto a outras divindades, em Israel Norte, estava em pleno curso. No entanto, a partir da análise conjunta da Estela de Dã, que tem como provável autor o rei Hazael de Damasco, somos desafiados a ler esta história pelas entrelinhas não contempladas pelo texto, que apontam para uma participação ativa de Hazael, nos desfechos referentes a Revolta de Jeú, como sendo o responsável direto que proporcionou a subida de Jeú ao trono em Israel, clarificando desta forma este importante período na história Bíblica. Para tal análise, observar-se-á três distintos tópicos, ligados diretamente ao tema proposto: (1) A Revolta de Jeú e a Redação Deuteronomista, a partir do estudo exegético da perícope de 2 Reis 10,28-36, onde estão descritas informações pontuais sobre período em que Jeú reinou em Israel; (2) Jeú e a Estela de Dã, a partir da apresentação e análise do conteúdo da Estela de Dã, tratando diretamente dos desdobramentos da guerra em Ramote de Gileade, de onde se dá o ponto de partida à Revolta de Jeú; e por fim (3) O Império da Síria, onde a partir da continuidade da análise do conteúdo da Estela de Dã, demonstraremos a significância deste reino, além de apontamentos diretamente ligados ao reinado de Hazael, personagem mui relevante no evento da Revolta de Jeú.

Towards a semiology of the periodic review of UK regulated utilities

This note explores the regulatory process of UK privatised utilities through the periodic review of prices. It provides a brief history of the privatisation programme in the UK and the theoretical arguments for the price-cap regulation that has been used. It argues that regulatory process appears to involve a covert dialogue and exchange of information between the regulator and regulated and also a second separate review process that consists of an overt dialogue. Using a semiotic analysis the authors suggest that the unfolding of each of these overt reviews follows a very similar pattern that is constantly being re-enacted. It is concluded that further research is required into the relative importance of the two separate review processes in the setting of the price-cap.

Regulation - the movie:a semiotic study of the periodic review of UK regulated industry

This paper explores the regulatory process of UK privatised utilities as manifest in the periodic review of prices. Two separate review processes are identified, operating concurrently - a covert dialogue between the regulator and the regulated and an overt dialogue taking place in the public arena. Using a semiotic analysis of the review the authors argue that the overt event is the real review. Furthermore they argue that the unfolding of each review is so similar that it can be likened to a film script which is constantly re-enacted. The purpose of the review as a legitimating vehicle for the regulator and regulated, who exist in a symbiotic relationship, is explored in terms of the semiotics involved and the myth creation role of legitimation in order to explain the significance of the regulatory process.

The interaction of fast neutrons with shielding and fusion blanket materials

In the present work the neutron emission spectra from a graphite cube, and from natural uranium, lithium fluoride, graphite, lead and steel slabs bombarded with 14.1 MeV neutrons were measured to test nuclear data and calculational methods for D - T fusion reactor neutronics. The neutron spectra measured were performed by an organic scintillator using a pulse shape discrimination technique based on a charge comparison method to reject the gamma rays counts. A computer programme was used to analyse the experimental data by the differentiation unfolding method. The 14.1 MeV neutron source was obtained from T(d,n)4He reaction by the bombardment of T - Ti target with a deuteron beam of energy 130 KeV. The total neutron yield was monitored by the associated particle method using a silicon surface barrier detector. The numerical calculations were performed using the one-dimensional discrete-ordinate neutron transport code ANISN with the ZZ-FEWG 1/ 31-1F cross section library. A computer programme based on Gaussian smoothing function was used to smooth the calculated data and to match the experimental data. There was general agreement between measured and calculated spectra for the range of materials studied. The ANISN calculations carried out with P3 - S8 calculations together with representation of the slab assemblies by a hollow sphere with no reflection at the internal boundary were adequate to model the experimental data and hence it appears that the cross section set is satisfactory and for the materials tested needs no modification in the range 14.1 MeV to 2 MeV. Also it would be possible to carry out a study on fusion reactor blankets, using cylindrical geometry and including a series of concentric cylindrical shells to represent the torus wall, possible neutron converter and breeder regions, and reflector and shielding regions.