Cycloviolacin H4, a hydrophobic cyclotide from Viola hederaceae

Cycloviolacin H4, a new macrocyclic miniprotein comprising 30 amino acid residues, was isolated from the underground parts of the Australian native violet Viola hederaceae. Its sequence, cyclo-(CAESCVWIPCTVTALLGCSCSNNVCYNGIP), was determined by nanospray tandem mass spectrometry and quantitative amino acid analysis. A knotted disuffide arrangement, which was designated as a cyclic cystine knot motif and characteristic to all known cyclotides, is proposed for stabilizing the molecular structure and folding. The cyclotide is classified in the bracelet subfamily of cyclotides due to the absence of a cis-Pro peptide bond in the circular peptide backbone. A model of its three-dimensional structure was derived based on the template of the homologous cyclotide vhr1 (Trabi et al. Plant Cell 2004, 16, 2204-2216). Cycloviolacin H4 exhibits the most potent hemolytic activity in cyclotides reported so far, and this activity correlates with the size of a surface-exposed hydrophobic patch. This work has thus provided insight into the factors that modulate the cytotoxic properties of cyclotides.

Discovery and characterization of a linear cyclotide from Viola odorata: Implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule. (c) 2006 Elsevier Ltd. All rights reserved.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice ( Oryza sativa), maize ( Zea mays), and wheat ( Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley ( Hordeum vulgare) have tissue-specific expression patterns.

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.

NMR of conotoxins: structural features and an analysis of chemical shifts of post-translationally modified amino acids

Conotoxins are small conformationally constrained peptides found in the venom of marine snails of the genus Conus. They are usually cysteine rich and frequently contain a high degree of post-translational modifications such as C-terminal amidation, hydroxylation, carboxylation, bromination, epimerisation and glycosylation. Here we review the role of NMR in determining the three-dimensional structures of conotoxins and also provide a compilation and analysis of H-1 and C-13 chemical shifts of post-translationally modified amino acids and compare them with data from common amino acids. This analysis provides a reference source for chemical shifts of post-translationally modified amino acids. Copyright (C) 2006 John Wiley & Sons, Ltd.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

The cyclotides and related macrocyclic peptides as scaffolds in drug design

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

The Pineapple EST sequencing and microarray project

We have initiated an EST sequencing project to survey a range of expressed sequences from green fruit, yellow fruit, roots, and root-knot nematode infected root/gall tissues. In total, 5681 edited EST sequences were retrieved. Clone redundancy was high in the fruit libraries, with the combined fruit 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of all fruit EST clone sequences clustered within approximately 14 and 9% of contigs from green unripe and yellow ripe libraries respectively, indicating that a small subset of genes dominates the majority of the transcriptome. The root and root/gall libraries had lower levels of redundancy than the fruit libraries. Half of the root/gall ESTs clustered within approximately 40% of all contigs, indicating the roots possess a more complex transcriptome. Contig assembly and cluster analysis revealed major differences in the abundant gene sequences expressed between the unripe green and the ripe yellow fruit tissues, or gene sequences expressed between the weeks 1-4 and weeks 5-10 nematode infected gall vascular cylinder libraries.

As inter-ações entre espaço do corpo e o movimento dançado

This dissertation seeks to reflect about the relations between danced movement and body space (Kinesphere), and their contributions to the expansion of the expressive possibilities of the subject on dancing. According to Rudolf Laban there is no space that is empty, because it is always being modified and signified at every moment by the movement. Space exists because we interact with it, at the same time movement occours configuring a signifcant space that is incessantly transformed. In this sense, space, body and movement appear in this research as interconnected and interdependent. For this discussion we have as main interlocutor the studies of Rudolf Laban. The nature of this research is qualitative and descriptive. This is a context that embraces the phenomenon of dance and as such it is based on a dimension that doesn't deal with mensurability, but with the art scene, fruitful in its infinite openness to the creation of multiple significances for what has been lived. We also propose to present a report about the practical study developed in the discipline Coreologia in the licentiate course of Dance in UFRN, as well as the analysis of the interviews applied to students of this curricular component. The questions were developed in a way that lead to a reflection about the experience of those interviewed in this discipline, thus generating material for us to discuss how the students perceive dance based on the relational study between space and movement. We realize that this study may favor an understanding of the relations that the experienced movement in the act of the dance weaves along the spatiality that receives and fills our bodies, resignifying the vision of a space which is restrict to the mere place were the body moves and occupies. It also favors the reflections concerning the body that moves and creates spatiality when dancing, thereby bringing to the Performing Arts a chance to think and to experience on the expansion of the expressive gesture in dance and beyond it, led by the recognition of the principles that organize human movement pointed by Laban. It also contributes on the formation of the students in licentiate courses of Dance by questioning the ways to appropriate the contents worked in a graduation discipline as regards to the availability of the body for dance. This dissertation is divided in three parts, called Impulsos. In the First Impulso: “Primeiros Gestos Textuais”, we find an introduction to concepts and ideas of body, movement and space that permeates all the work. In the Second Impulso: "Nós", the triad body-space-motion is discussed using the metaphorical image of a knot that binds these three concepts. The third and final Impulso: "Enlaces" deal with impressions and discoveries lived during the experimentation of the principles of inter-actions studied here, in the lessons of the already mentioned discipline

Highly sensitive refractive index sensor based on cascaded microfiber knots with Vernier effect

We propose and experimentally demonstrate a refractive index (RI) sensor based on cascaded microfiber knot resonators (CMKRs) with Vernier effect. Deriving from high proportional evanescent field of microfiber and spectrum magnification function of Vernier effect, the RI sensor shows high sensitivity as well as high detection resolution. By using the method named "Drawing-Knotting-Assembling (DKA)", a compact CMKRs is fabricated for experimental demonstration. With the assistance of Lorentz fitting algorithm on the transmission spectrum, sensitivity of 6523nm/RIU and detection resolution up to 1.533 x 10-7 RIU are obtained in the experiment which show good agreement with the numerical simulation. The proposed all-fiber RI sensor with high sensitivity, compact size and low cost can be widely used for chemical and biological detection, as well as the electronic/magnetic field measurement

Augmentations and Rulings of Legendrian Links

For any Legendrian knot in R^3 with the standard contact structure, we show that the existence of an augmentation to any field of the Chekanov-Eliashberg differential graded algebra over Z[t,t^{-1}] is equivalent to the existence of a normal ruling of the front diagram, generalizing results of Fuchs, Ishkhanov, and Sabloff. We also show that any even graded augmentation must send t to -1.

We extend the definition of a normal ruling from J^1(S^1) given by Lavrov and Rutherford to a normal ruling for Legendrian links in #^k(S^1\times S^2). We then show that for Legendrian links in J^1(S^1) and #^k(S^1\times S^2), the existence of an augmentation to any field of the Chekanov-Eliashberg differential graded algebra over Z[t,t^{-1}] is equivalent to the existence of a normal ruling of the front diagram. For Legendrian knots, we also show that any even graded augmentation must send t to -1. We use the correspondence to give nonvanishing results for the symplectic homology of certain Weinstein 4-manifolds.

Histoire évolutive de la galaxie spirale NGC 5430

La galaxie spirale barrée NGC 5430 est particulière en ce sens qu’elle présente un noeud Wolf-Rayet très lumineux et des bras asymétriques. Des spectres longue-fente le long de la barre et dans le bras déformé ainsi que des données SpIOMM couvrant l’ensemble de la galaxie ont été analysées. L’absorption stellaire sous-jacente a été soustraite des spectres longue-fente à l’aide d’un ajustement de modèles théoriques de populations stellaires fait avec le programme GANDALF. L’absorption a un impact très important sur le calcul de l’extinction ainsi que sur les différents diagnostics propres aux régions HII et aux populations stellaires jeunes. Enfin, cette étude montre que NGC 5430 comporte une composante gazeuse ionisée diffuse sur toute son étendue et qu’il est important d’en tenir compte afin d’appliquer correctement les diagnostics. Un des scénarios évolutifs proposés au terme de cette étude est que le noeud Wolf-Rayet constitue le restant d’une petite galaxie ou d’un nuage intergalactique qui serait entré en collision avec NGC 5430. Une structure englobant le noeud Wolf-Rayet se déplace à une vitesse considérablement inférieure (50 - 70 km s-1) à celle attendue à une telle distance du centre de la galaxie (200 - 220 km s-1). De plus, le noeud Wolf-Rayet semble très massif puisque l’intensité maximale du continu stellaire de cette région est semblable à celle du noyau et est de loin supérieure à celle de l’autre côté de la barre. Le nombre d’étoiles Wolf-Rayet (2150) est aussi considérable. Il n’est toutefois pas exclu que la différence de vitesses observée témoigne d’un écoulement de gaz le long de la barre, qui alimenterait la formation stellaire du noeud Wolf-Rayet ou du noyau.

Encountering the Spectre of Painting

When thinking what paintings are, I am continually brought back to my memory of a short sequence in Alfred Hitchcock’s Vertigo. In the scene, Kim Novak’s Madeleine is seated on a bench in an art gallery. She is apparently transfixed by a painting, Portrait of Carlotta. Alongside James Stewart, we watch her looking intently. Madeleine is pretending to be a ghost. At this stage she does not expect us to believe she is a ghost, but simply to immerse ourselves in the conceit, to delight in the shudder. Madeleine’s back is turned away from us, and as the camera draws near to show that the knot pattern in her hair mirrors the image in the portrait, I imagine Madeleine suppressing a smile. She resolutely shows us her back, though, so her feint is not betrayed. Madeleine’s stillness in this scene makes her appear as an object, a thing in the world, a rock or a pile of logs perhaps. We are not looking at that thing, however, but rather a residual image of something creaturely, a spectre. This after-image is held to the ground both by the gravity suggested by its manifestation and by the fine lie - the camouflage - of pretending to be a ghost. Encountering a painting is like meeting Madeleine. It sits in front of its own picture, gazing at it. Despite being motionless and having its back to us, there is a lurching sensation the painting brings about by pretending to be the ghost of its picture, and, at the same time, never really anticipating your credulity.

Successful management of evisceration occurred after exploratory laparotomy for bilateral ovarian micropapillary serous borderline tumors

Micropapillary serous borderline tumor of the ovary is characterized by a more frequent association with extraovarian, especially invasive, implants. The aim of this study was to report the clinicopathological findings of a rare case of micropapillary serous borderline tumor of the ovary since there are less than 100 similar cases in the published literature. Additionally, the successful management of evisceration that complicated the postoperative stay of the patient is analyzed. The incidence of this severe complication is estimated between 0.29-2.3%. There are four main causes: suture tearing through the fascia, knot failure, suture failure, and extrusion of abdominal contents between sutures placed too far apart. At least 50% of the cases are due to technical error with a potentially lethal result.