Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cycloticles or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata Ell as well as three other clones for related cycloticles from the African plant Olden-landia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cycloticles have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

Do generalized visual training programmes for sport really work? An experimental investigation

We assessed the effectiveness of two generalized visual training programmes in enhancing visual and motor performance for racquet sports. Forty young participants were assigned equally to groups undertaking visual training using Revien and Gabor's Sports Vision programme (Group 1), visual training using Revien's Eyerobics (Group 2), a placebo condition involving reading (Group 3) and a control condition involving physical practice only (Group 4). Measures of basic visual function and of sport-specific motor performance were obtained from all participants before and immediately after a 4-week training period. Significant pre- to post-training differences were evident on some of the measures; however, these were not group-dependent. Contrary to the claims made by proponents of generalized visual training, we found no evidence that the visual training programmes led to improvements in either vision or motor performance above and beyond those resulting simply from test familiarity.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Carbene and nitrene rearrangements: A theoretical study of cyclic allenes and carbenes, carbodiimides, and azirines

B3LYP/6-31G(d) calculations of structures, energies, and infrared spectra of several rearrangement products of (hetero)aromatic nitrenes and carbenes are reported. 3-Isoquinolylnitrene 36 ring closes to the azirine 37 prior to ring expansion to the potentially stable but unobserved seven-membered-ring carbodiimide 38 and diazacycloheptatrienylidene C-s-39S. A new, stable cycloheptatrienylidene, C-s-19S, is located on the naphthylcarbene energy surface. 4-Quinolylnitrene undergoes reaction via the azirine 50 in solution, but ring expansion to the stable seven-membered-ring ketenimine 47 under Ar matrix photolysis conditions. There is excellent agreement between calculated infrared spectra of 1,5-diazacyclohepta-1,2,4,6-tetraene 54 (obtained by photolysis of 4-pyridyl azide), 1-azacyclohepta-1,2,4,6-tetraene 5, 1-azacyclohepta-1,3,5,6-tetraene 55, and 1-azacyclohepta-1,3,4,6-tetraene 56 and the available experimental data.

Use of cyclic variation of integrated backscatter to assess contractile reserve and myocardial viability in chronic ischemic left ventricular dysfunction

The detection of viable myocardium has important implications for management, but use of stress echocardiography to detect this is subjective and requires exposure to dobutamine. We investigated whether cyclic variation (CV) of integrated backscatter (IB) from the apical views could provide a resting study for detection of contractile reserve (CR) and prediction of myocardial viability in 27 patients with chronic ischemic left ventricular (LV) dysfunction. Repeat echocardiography was performed after 6.7 +/- 3.8 months of follow-up; 14 patients underwent revascularization and 13 were treated medically. Using a standardized dobutamine echocardiography (DbE) protocol, images from three apical views were acquired at 80-120 frames/sec at rest and during stress. CR was identified if improvement of wall motion was observed at low dose (5 or 10 mug/kg/min) DbE. Myocardial viability was characterized by improvement at follow-up echocardiography in patients with revascularization. CVIB at rest and low dose dobutamine were assessed in 194 segments with resting asynergy (severe hypokinesis or akinesis), of which 88 (45%) were in patients who underwent revascularization. Of these, CVIB could be measured in 190 (98%) segments at rest and 185 (95%) at low dose dobutamine. Sixty-two (33%) segments had CR during low dose DbE and 50 (57%) segments showed wall-motion recovery (myocardial viability) at follow-up echocardiography. Segments with CR had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P = 0.005) than segments without CR. Using optimal thresholds of CVIB (> 8.2 dB) at rest, the accuracy of CVIB for detecting CR was 70%. Compared with nonviable segments, viable segments had significantly higher CVIB at rest (P < 0.001) and low dose dobutamine (P < 0.001). Using optimal thresholds of CVIB (> 5.3 dB) at rest, the accuracy of CVIB for detecting myocardial viability was 85%, which was higher than that in conventional DbE (62%, P < 0.01). Thus, assessment of CV.TB from the apical views is a feasible and accurate tool for detecting CR and predicting myocardial viability in chronic LV dysfunction.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

A subset-oriented algorithm for minimizing the number of steps required for synthesis of cyclic-peptide libraries

Libraries of cyclic peptides are being synthesized using combinatorial chemistry for high throughput screening in the drug discovery process. This paper describes the min_syn_steps.cpp program (available at http://www.imb.uq.edu.au/groups/smythe/tran), which after inputting a list of cyclic peptides to be synthesized, removes cyclic redundant sequences and calculates synthetic strategies which minimize the synthetic steps as well as the reagent requirements. The synthetic steps and reagent requirements could be minimized by finding common subsets within the sequences for block synthesis. Since a brute-force approach to search for optimum synthetic strategies is impractically large, a subset-orientated approach is utilized here to limit the size of the search. (C) 2002 Elsevier Science Ltd. All rights reserved.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Non-compact generalized variational inequalities for quasi-monotone and hemi-continuous operators with applications

Some results are obtained for non-compact cases in topological vector spaces for the existence problem of solutions for some set-valued variational inequalities with quasi-monotone and lower hemi-continuous operators, and with quasi-semi-monotone and upper hemi-continuous operators. Some applications are given in non-reflexive Banach spaces for these existence problems of solutions and for perturbation problems for these set-valued variational inequalities with quasi-monotone and quasi-semi-monotone operators.

Explicit/implicit partitioning and a new explicit form of the generalized alpha method

Most finite element packages use the Newmark algorithm for time integration of structural dynamics. Various algorithms have been proposed to better optimize the high frequency dissipation of this algorithm. Hulbert and Chung proposed both implicit and explicit forms of the generalized alpha method. The algorithms optimize high frequency dissipation effectively, and despite recent work on algorithms that possess momentum conserving/energy dissipative properties in a non-linear context, the generalized alpha method remains an efficient way to solve many problems, especially with adaptive timestep control. However, the implicit and explicit algorithms use incompatible parameter sets and cannot be used together in a spatial partition, whereas this can be done for the Newmark algorithm, as Hughes and Liu demonstrated, and for the HHT-alpha algorithm developed from it. The present paper shows that the explicit generalized alpha method can be rewritten so that it becomes compatible with the implicit form. All four algorithmic parameters can be matched between the explicit and implicit forms. An element interface between implicit and explicit partitions can then be used, analogous to that devised by Hughes and Liu to extend the Newmark method. The stability of the explicit/implicit algorithm is examined in a linear context and found to exceed that of the explicit partition. The element partition is significantly less dissipative of intermediate frequencies than one using the HHT-alpha method. The explicit algorithm can also be rewritten so that the discrete equation of motion evaluates forces from displacements and velocities found at the predicted mid-point of a cycle. Copyright (C) 2003 John Wiley Sons, Ltd.

Integrable generalized spin ladder models based on the SU(1|3) and SU(3|1) algebras

We present two integrable spin ladder models which possess a general free parameter besides the rung coupling J. The models are exactly solvable by means of the Bethe ansatz method and we present the Bethe ansatz equations. We analyze the elementary excitations of the models which reveal the existence of a gap for both models that depends on the free parameter. (C) 2003 American Institute of Physics.

Estudo da corrosão dos aços AISI 1010 e AISI 316L em soluções com elevado teor de cloreto e em ácido naftênico

A mudança no mercado global do petróleo nos últimos anos, com o declínio das reservas de óleo leve, têm forçado a busca por novos campos petrolíferos em ambientes mais remotos, como nos campos localizados na camada pré-sal, e a exploração de óleos pesados que possuem elevado teor de ácidos naftênicos. Isso acarreta em grandes desafios para a previsão do desempenho de materiais frente às novas condições ambientais em que estão inseridos. No presente trabalho, o comportamento da corrosão do aço carbono AISI 1010 e do aço inoxidável AISI 316L foi estudado em soluções aquosas com elevado teor de cloreto e em solução de ácido naftênico ciclopentanóico a fim de ter melhor entendimento da ação dessas espécies no processo de corrosão e simular a corrosão pela água de produção na indústria petrolífera. Foram aplicadas as técnicas de potencial de circuito aberto, polarização potenciodinâmica, voltametria cíclica, espectroscopia de impedância eletroquímica, espectroscopia Raman, microscopia eletrônica de varredura e microscopia de força atômica, usadas, em cada caso, de acordo com a conveniência. O aumento da [Cl-] na faixa de 1,2–2,8 mol.L-1 não altera os processos catódicos e anódicos perto do Ecorr para os aços AISI 1010 e AISI 316L. Em condições de sobrepotenciais afastados do Ecorr, o aumento de [Cl-] aumenta os processos oxidativos de corrosão, o que é expresso pelas maiores densidades de corrente e carga anódica e aumento da perda de massa sofrida pelos eletrodos de ambos os aços. Portanto, os danos da corrosão são mais intensos quando se aumenta a [Cl-]. O aço AISI 1010 é ativo nas soluções de NaCl e a corrosão se propaga livremente de forma uniforme. Para o aço AISI 316L, uma ampla faixa de passividade pode ser vista nas soluções de NaCl; no Epit ocorre a ruptura do filme passivo e o crescimento de pites estáveis. Após 24 h de imersão em soluções de sulfato de sódio (branco) e de ácido naftênico ciclopentanóico ocorre crescimento de filme de óxido e as fases α-Fe2O3, Fe3O4 e δ-FeO(OH) foram identificadas nos espécimes de aço AISI 1010 e Fe3O4 foi identificado nos defeitos do filme prévio presente na superfície do aço AISI 316L. Os filmes formados em solução de ácido ciclopentanóico possuem menor resistência à polarização, maior rugosidade e maior taxa de corrosão quando comparado aos filmes crescidos na solução branco, para ambos os aços. A presença do ácido naftênico muda a forma como a reação de corrosão se procede e contribui para o aumento da corrosão. A corrosão naftênica foi mais pronunciada no aço carbono porque a presença dos elementos de liga no aço inox reduzem o número de sítios ativos ricos em Fe e tornam menos oportuna a ligação do Fe com o naftenato.