The structural and functional diversity of naturally occurring antimicrobial peptides

Antimicrobial peptides occur in a diverse range of organisms from microorganisms to insects, plants and animals. Although they all have the common function of inhibiting or killing invading microorganisms they achieve this function using an extremely diverse range of structural motifs. Their sizes range from approximately 10-90 amino acids. Most carry an overall positive charge, reflecting a preferred mode of electrostatic interaction with negatively charged microbial membranes. This article describes the structural diversity of a representative set of antimicrobial peptides divided into five structural classes: those with agr-helical structure, those with bgr-sheet structure, those with mixed helical / bgr- sheet structure, those with irregular structure, and those incorporating a macrocyclic structure. There is a significant diversity in both the size and charge of molecules within each of these classes and between the classes. The common feature of their three-dimensional structures is, however, that they have a degree of amphipathic character in which there is separate localisation of hydrophobic regions and positively charged regions. An emerging trend amongst antimicrobial proteins is the discovery of more macrocyclic analogues. Cyclisation appears to impart an additional degree of stability on these molecules and minimizes proteolytic cleavage. In conclusion, there appear to be a number of promising opportunities for the development of novel clinically useful antimicrobial peptides based on knowledge of the structures of naturally occurring antimicrobial molecules.

Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.

The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an a-helix and a triple-stranded anti-parallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized up motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure - activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP. (C) 2002 Elsevier Science Ltd. All rights reserved.

Circular proteins - no end in sight

Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold

The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterised proteins and two proteins that lack methyltransferase activity.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Respiratory health effects of cannabis: Position statement of the Thoracic Society of Australia and New Zealand

Both the gaseous and the particulate phases of tobacco and cannabis smoke contain a similar range of harmful chemicals. However, differing patterns of inhalation mean that smoking a 'joint' of cannabis results in exposure to significantly greater amounts of combusted material than with a tobacco cigarette. The histopathological effects of cannabis smoke exposure include changes consistent with acute and chronic bronchitis. Cellular dysplasia has also been observed, suggesting that, like tobacco smoke, cannabis exposure has the potential to cause malignancy. These features are consistent with the clinical presentation. Symptoms of cough and early morning sputum production are common (20-25%) even in young individuals who smoke cannabis alone. Almost all studies indicate that the effects of cannabis and tobacco smoking are additive and independent. Public health education should dispel the myth that cannabis smoking is relatively safe by highlighting that the adverse respiratory effects of smoking cannabis are similar to those of smoking tobacco, even although it remains to be confirmed that smoking cannabis alone leads to the development of chronic lung disease.

Growth of papaya nodal and rooted shoot cultures as affected by ACC, STS, culture vessel size and incubation conditions

Nodal shoot cultures of 'Clone 003', a selected Australian papaya cultivar, were cultured on modified De Fossard medium supplemented with chemicals that either promote ethylene evolution or inhibit action while in culture. Nodal shoot cultures grown in the presence of 1-aminocyclopropane carboxylic acid (ACC, 1.0 mM) resulted in a significant reduction in percent fresh and dry weights, shoot length, leaf area, petiole length and chlorophyll content, but leaf development was significantly increased. In contrast, nodal cultures grown in the presence of silver thiosulphate (STS, 0.5 mM) significantly produced the highest percentage of fresh and dry weights, shoot length, leaf production, leaf area expansion, petiole length and leaf chlorophyll content. Nodal cultures and rooted whole plantlets placed in medium-sized (125 mL) culture vessels had significantly better growth than those cultures placed in small (70 mL) or in large (250 mL) vessels. Cultures grown in medium-sized vessels had higher fresh and dry weights, longer shoots, more leaves and larger leaf area than those cultures placed in smaller or larger vessels. Similarly, values for said growth parameters and for chlorophyll content of the nodal and rooted whole plantlets were higher when they were incubated under high light intensity of 120 mumol m(-2)s(-1) at a prevailing temperature of either 20+/-1 C or 25+/-1 C.

Sensitivity of Helicoverpa armigera nucleopolyhedrovirus polyhedra to sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

A new wavelet-based adaptive method for solving population balance equations

A new wavelet-based adaptive framework for solving population balance equations (PBEs) is proposed in this work. The technique is general, powerful and efficient without the need for prior assumptions about the characteristics of the processes. Because there are steeply varying number densities across a size range, a new strategy is developed to select the optimal order of resolution and the collocation points based on an interpolating wavelet transform (IWT). The proposed technique has been tested for size-independent agglomeration, agglomeration with a linear summation kernel and agglomeration with a nonlinear kernel. In all cases, the predicted and analytical particle size distributions (PSDs) are in excellent agreement. Further work on the solution of the general population balance equations with nucleation, growth and agglomeration and the solution of steady-state population balance equations will be presented in this framework. (C) 2002 Elsevier Science B.V. All rights reserved.

Resistência natural da madeira de sete espécies de eucalipto ao apodrecimento

A resistência natural ao apodrecimento se constitui numa importante propriedade da madeira. Em se tratando das madeiras provenientes do gênero Eucalyptus, tal atributo se torna ainda mais importante, uma vez que tais madeiras são, na grande maioria das vezes, de difícil tratabilidade pelos métodos de tratamento convencionais. O objetivo do presente trabalho foi determinar a resistência natural da madeira de sete espécies de eucalipto em ensaio acelerado de laboratório ao fungo causador da podridão-parda Gloeophyllum trabeum. Foram ensaiadas madeiras das espécies de Corymbia citriodora, Eucalyptus tereticornis, E. paniculata, E. pilularis, E. cloeziana, E. urophylla e E. grandis com 16 anos de idade, que cresceram na Estação Experimental de Anhembi, em São Paulo, pertencente ao Departamento de Ciências Florestais da ESALQ/USP. No que diz respeito ao apodrecimento causado pelo fungo Gloeophyllum trabeum, a madeira de eucalipto das sete espécies pode ser considerada resistente, uma vez que os valores de perda de massa foram sempre inferiores a 10%.

Imagens da escola como mediadoras do processo formativo dos jovens no ensino da arte: diálogos com a história, memória e ambientes intraescolares

Esta pesquisa apresenta as imagens da escola como mediadoras do processo formativo dos jovens no ensino da Arte em diálogo com a história, memória e ambientes intraescolares. Inscreve-se no debate produzido pela linha de pesquisa em Educação e Linguagens. Analisa a formação dos jovens numa turma do terceiro ano do Ensino Médio na Escola Estadual de Ensino Médio “Hunney Everest Piovesan”, localizada no município de Cariacica no Estado do Espírito Santo. O estudo foi realizado em 2013 e tem como objetivo analisar as imagens escolares como mediadoras na formação dos estudantes do Ensino Médio no ensino da Arte em diálogo com a história, memória e ambientes intraescolares. Ao mesmo tempo, por meio de trabalho colaborativo, contribui para (re)construção da história da instituição, significando-a junto aos alunos e comunidade escolar. Por meio de intervenção artística com imagens da escola propõe reflexão crítica, analisando a formação dos jovens no terceiro ano do Ensino Médio. Para compreender os conceitos de mediação e meio social, estabelece um diálogo com as obras de Lev Semenovith Vigotski. A partir dos estudos de Maria Ciavatta e Schütz–Foerste procura entender a mediação imagética e sua dimensão educativa. Dialoga ainda com Frago, Escolano e Buffa, ampliando as reflexões sobre os ambientes intraescolares, dimensionando a imagética desses espaços e o senso de pertencimento à escola a partir da impregnação pela história e memória. A investigação pauta-se nos referenciais do método qualitativo e colaborativo de pesquisa. A produção dos dados contou com o recolhimento e análise documental de um conjunto de fontes primárias e secundárias formadas por livro de registro de funcionários da escola da década de 1970, diários de classe, recortes de jornais, convites de formaturas e registros fotográficos escolares dessa mesma década até a atualidade. Além desses documentos históricos, foram analisadas entrevistas de antigos alunos e questionários dos alunos do terceiro ano do Ensino Médio da instituição. Relata brevemente experiências de curto intercâmbio acadêmico realizado na Universidade de Ancara, na Turquia, com o objetivo de ampliar os horizontes de referência sociocultural, em especial com o contexto educacional do Ensino Médio euroasiático, identificando os espaços de formação dos jovens no contexto turco e suas relações com a educação brasileira. Esta experiência apresentada não integra a análise desta dissertação, mas projeta a discussão para novos estudos. A análise se elabora a partir da triangulação, entre outros, do referencial teórico com o processo de intervenção, produção de dados e no debate acadêmico em diferentes contextos. A partir da caminhada acadêmica permeada pela história e memória, inferimos que as imagens escolares medeiam o processo formativo dos jovens do terceiro ano do Ensino Médio no ensino da Arte. Contribuem para o cultivo da memória escolar, enquanto presença impregnada pela história da instituição, por meio da imagética dos seus ambientes intraescolares. Constatamos ainda que as imagens colaboram para o (re)conhecimento por parte desses sujeitos do seu papel ativo, autônomo e transformador da realidade escolar na qual estão inseridos.

Pensando o currículo com as crianças: ou sobre aprendizagens inventivas na educação infantil

Ninguém consegue sair ileso de um encontro com o currículo e com a escola, principalmente diante de relações tão assimétricas de poder que não valorizam o que as crianças têm a dizer. Quantas narrativas curriculares sobre as crianças? Quantas narrativas curriculares com as crianças? As narrativas e as fotografias se seguirão nesta pesquisa, porque com elas nos colocamos a pensar nos processos de criação curricular, explorando novas sensibilidades, novas maneiras de ver e falar, a partir das redes de sentidos, forjadas nos contextos vividos, imaginados ou pensados, dentrofora da escola. Também incluímos a forma como as crianças se relacionam com as práticas pedagógicas dos professores e do modo como se relacionam com a escola. Nessa perspectiva, interessa-nos pensar em como os usos (CERTEAU, 1994) da imagem fotográfica são capazes de afetar (e até transformar) as práticas curriculares, traçando algo da potência da/na imagem fotográfica em sua função fabuladora por meio das oficinas de fotografias realizadas com as crianças em virtude da pesquisa. A fotografia se torna potente como um recurso para provocar a invenção tessitura de outros sentidos em currículo não porque em sua materialidade ela está repleta de sentidos de currículo à priori, mas porque pode ou não, ao ser usada (CERTEAU, 1994), ao ser vista pelas crianças, agenciar outros possíveis para o currículo. Assim, o foco da discussão não é a fotografia em si nem a criança em si, ou seja, não há protagonismo nem da criança nem da fotografia. O foco está nas relações, naquilo que nos passa, isto é, na experiência estética (LARROSA, 2004b) que ocorre ao entrarmos em contato, ao vermos, ao compormos com as fotografias! Nesse sentido, pensamos as oficinas de fotografias como um dispositivo de criação e produção de acontecimentos em currículo, considerando-as máquinas de fazer “ver e falar”, o que as justifica como uma estratégia narrativa capaz de produzir acontecimentos na imagem e no mundo. Que sentidos de currículo são produzidos em multiplicidades? Pelas minoridades pretendemos movimentar nosso pensamento: que quer pensar um currículo como fabulação sem dizer o que ele é, mas no que ele vai se transformando com a chegada das crianças. No encontro das imagens com as palavras, em que o currículo vai se transformando? Sob a mesma superfície chamada currículo em extensão com as crianças, co-habitantes, encontrar os modos de olhar esse currículo e de dizê-lo por meio das fotografias, das narrativas, dos cartazes, dos desenhos, das poesias... As conversas com as crianças provocam o real, colocam em desequilíbrio algumas ideias feitas em educação, exigindo reordenações e invenções de outros pensamentos para a educação. É dessa criação de efeitos impensáveis que surge a invenção de currículos possíveis. Aprender a olhar mais (até cansar!) aquilo que não percebemos no dia a dia tem uma dimensão política muito importante; por meio desse gesto (que aprendemos com as crianças) podemos criar um novo pensamento político em educação. A partir daquilo que nos dá a ver, as crianças vão inaugurar sentidos impertinentes, desestabilizadores daquilo que chamamos de currículo e escola. O desafio consiste em falar da força contida na imagem fotográfica sem vontade de interpretá-la ou descrevê-la, mas escrever e pensar pelas fotografias num movimento de criação de sentidos e acontecer por elas.