Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

A yield architecture framework to explain adaptation of pearl millet to environmental stress

Functional knowledge of the physiological basis of crop adaptation to stress is a prerequisite for exploiting specific adaptation to stress environments in breeding programs. This paper presents an analysis of yield components for pearl millet, to explain the specific adaptation of local landraces to stress environments in Rajasthan, India. Six genotypes, ranging from high-tillering traditional landraces to low-tillering open-pollinated modern cultivars, were grown in 20 experiments, covering a range of nonstress and drought stress patterns. In each experiment, yield components (particle number, grain number, 100 grain mass) were measured separately for main shoots, basal tillers, and nodal tillers. Under optimum conditions, landraces had a significantly lower grain yield than the cultivars, but no significant differences were observed at yield levels around 1 ton ha(-1). This genotype x environment interaction for grain yield was due to a difference in yield strategy, where landraces aimed at minimising the risk of a crop failure under stress conditions, and modem cultivars aimed at maximising yield potential under optimum conditions. A key aspect of the adaptation of landraces was the small size of the main shoot panicle, as it minimised (1) the loss of productive tillers during stem elongation; (2) the delay in anthesis if mid-season drought occurs; and (3) the reduction in panicle productivity of the basal tillers under stress. In addition, a low investment in structural panicle weight, relative to vegetative crop growth rate, promoted the production of nodal tillers, providing a mechanism to compensate for reduced basal tiller productivity if stress occurred around anthesis. A low maximum 100 grain mass also ensured individual grain mass was little affected by environmental conditions. The strategy of the high-tillering landraces carries a yield penalty under optimum conditions, but is expected to minimise the risk of a crop failure, particularly if mid-season drought stress occurs. The yield architecture of low-tillering varieties, by contrast, will be suited to end-of-season drought stress, provided anthesis is early. Application of the above adaptation mechanisms into a breeding program could enable the identification of plant types that match the prevalent stress patterns in the target environments. (C) 2003 E.J. van Oosterom. Published by Elsevier Science B.V. All rights reserved.

Preparation and characterization of Nickel-and cobalt-doped magnetites

Trabalho apresentado no I Simpósio Mineiro de Ciências dos Materiais, Ouro Preto, Novembro de 2001.

Characterization of the types of the Neotropical Pseudisobrachium (Hymenoptera: Bethylidae), with a key to species

Atualmente é difícil reconhecer a identidade de muitas espécies neotropicais de Pseudisobrachium Kieffer, 1904, principalmente por que as descrições e ilustrações disponíveis não são suficientes para permitir identificações precisas. Para resolver este problema, foram examinadas 115 espécies válidas, além de seus sinônimos juniores. Foram realizados doze atos nomenclaturais, e reconhecidas 110 espécies válidas para a região Neotropical. Foram designados dois lectótipos: Pristocera crassicornis Westwood and Pristocera haemorrhoidalis Westwood. Foram propostas sete sinonímias novas para espécies: Pseudisobrachium retusum Evans syn. nov. de P. pauxillum Evans; P. cunco Perez syn. nov. de P. erythrocephalum Evans; P. navajo Evans, P. rectangulatum Evans, P. emarginatum Evans e P. foutsi Evans syn. nov. de P. flavinervis Fouts; P. acuminatum Waichert & Azevedo syn. nov. de P. latum Waichert & Azevedo. Foi proposta a seguinte sinonímia nova para gênero: Parisobrachium Kieffer syn. nov. de Dissomphalus Ashmead. Foi estabelecida a seguinte combinação nova e revalidado o nome: Dissomphalus albipes (Kieffer) comb. nov. e nom. rev. de Pseudisobrachium paraguayense Kieffer.

Ácidos húmicos extraídos do lodo de esgoto sanitário e seus efeitos em plantas

O lodo de esgoto possui alto teor de matéria orgânica porém, também estão presentes diferentes poluentes e patógenos. Desta forma, neste trabalho foi extraído ácido húmico (AH) o qual é um composto resultante do fracionamento de substâncias húmicas que compreendem um grupo de compostos de carbono gerados na decomposição de resíduos orgânicos que sofrem ressíntese formando o húmus. O ácido húmico promove diversos benefícios nos vegetais, como crescimento, elongação celular, tolerância a estresses e aumento da permeabilidade da membrana plasmática. Com base nestas características, o presente trabalho teve como objetivo avaliar os efeitos citogenéticos (ensaio Allium cepa), fisiológicos, anatômicos e bioquímicos do ácido húmico do lodo de esgoto sanitário. Foi realizada caracterização elementar do material para a definição das doses. Após 20 dias de tratamento, foram realizadas coletas do material e, posteriormente, analisadas. A caracterização química do AH indicou-o como bom condicionador para culturas apresentando elevadas taxas de C, H e N. Não foi observado efeito de toxicidade, citotoxicidade, genotoxicidade e mutagenicidade do AH. Foi verificado aumento expressivo de todos os pigmentos fotossintéticos vegetais nas concentrações mais altas (2 mM C L-1 e 4 mM C L-1). Houve aumento da expressão da ATPAse em todos os tratamentos e das enzimas do estresse oxidativo (CAT, SOD, APX, GST) em diferentes concentrações. A integração destas análises permitiu concluir que o ácido húmico do lodo de esgoto pode ser utilizado como adubo orgânico, pois foi observado benefícios no vegetal tais como maior crescimento vegetal e aumento da atividade nas enzimas do estresse oxidativo.

Brazil and the economic, political, and environmental multilateralism: the Lula years (2003-2010)

This article discusses Brazil's multilateral policy under Luiz Inácio Lula da Silva's administration, highlighting economic, political and environmental themes. The main argument is that Brazil's multilateral relations during this government reached a new significance with the reinforcement of international coalitions and the articulation with the country's new development model. The country has been using multilateral forums as a way to achieve international projection and support for its development project, highlighting bargaining aspects, variable geometry coalitions and the strengthening of South-South cooperation

The G-77, BASIC, and global climate governance: a new era in multilateral environmental negotiations

The G-77 has historically organized the participation of developing countries in multilateral environmental negotiations. This article analyses the impact of a new coalition of emerging powers - Brazil, China, India, and South Africa as BASIC - on the G-77's role in climate governance. While there are important benefits for both sides in their relationship, I argue that the G-77 is also disadvantaged in several concrete ways by the BASIC countries.

Environmental issues and international relations, a new global (dis)order - the role of International Relations in promoting a concerted international system

Is it possible to talk about the rise of a new global (dis)order founded on the challenges posed by environmental issues? Through the review of the state of the art on the subject, this article analyzes the growing importance of the environment, and natural resources in particular, in international relations; and aims to raise awareness among International Relations scholars to the potential positive impact of the development of the discipline in integration with global environmental change studies.

Integrating environmental and economic life cycle analysis in product development: a material selection case study

Purpose Achieving sustainability by rethinking products, services and strategies is an enormous challenge currently laid upon the economic sector, in which materials selection plays a critical role. In this context, the present work describes an environmental and economic life cycle analysis of a structural product, comparing two possible material alternatives. The product chosen is a storage tank, presently manufactured in stainless steel (SST) or in a glass fibre reinforced polymer composite (CST). The overall goal of the study is to identify environmental and economic strong and weak points related to the life cycle of the two material alternatives. The consequential win-win or trade-off situations will be identified via a Life Cycle Assessment/Life Cycle Costing (LCA/LCC) integrated model. Methods The LCA/LCC integrated model used consists in applying the LCA methodology to the product system, incorporating, in parallel, its results into the LCC study, namely those of the Life Cycle Inventory (LCI) and the Life Cycle Impact Assessment (LCIA). Results In both the SST and CST systems the most significant life cycle phase is the raw materials production, in which the most significant environmental burdens correspond to the Fossil fuels and Respiratory inorganics categories. The LCA/LCC integrated analysis shows that the CST has globally a preferable environmental and economic profile, as its impacts are lower than those of the SST in all life cycle stages. Both the internal and external costs are lower, the former resulting mainly from the composite material being significantly less expensive than stainless steel. This therefore represents a full win-win situation. As a consequence, the study clearly indicates that using a thermoset composite material to manufacture storage tanks is environmentally and economically desirable. However, it was also evident that the environmental performance of the CST could be improved by altering its End-of-Life stage. Conclusions The results of the present work provide enlightening insights into the synergies between the environmental and the economic performance of a structural product made with alternative materials. Further, they provide conclusive evidence to support the integration of environmental and economic life cycle analysis in the product development processes of a manufacturing company, or in some cases even in its procurement practices.

The quest for a sustainable product: An environmental study of tyre recyclates

The main objective of the present study is to assess the environmental advantages of substituting aluminium for a polymer composite in the manufacture of a structural product (a frame to be used as a support for solar panels). The composite was made of polypropylene and a recycled tyres’ rubber granulate. Analysis of different composite formulations was performed, to assess the variation of the environmental impact with the percentage of rubber granulate incorporation. The results demonstrate that the decision on which of the two systems (aluminium or composite) has the best life cycle performance is strongly dependent on the End-of Life (EoL) stage of the composite frame. When the EoL is deposition in a landfill, the aluminium frame performs globally better than its composite counterpart. However, when it is incineration with energy recovery or recycling, the composite frame is environmentally preferable. The raw material production stage was found to be responsible for most of the impacts in the two frame systems. In that context, it was shown that various benefits can accrue in several environmental impact categories by recycling rubber tyres and using the resulting materials. This is in a significant part also due to the recycling of the steel in the tyres. The present work illustrates how it is possible to minimize the overall environmental impact of consumer products through the adequate selection of their constitutive materials in the design stage. Additionally it demonstrates how an adequate EoL planning can be an important issue when developing a sustainable product, since it can highly influence its overall life cycle performance.

Improving the environmental performance of bedding products by using life cycle assessment at the design stage

Improvement of the environmental performance of processes and products is a common objective in industry, and has been receiving increased attention in recent years. The main objective of this work is to evaluate the potential environmental impact of two bedding products, a polyurethane foam mattress (PFM) and a pocket spring mattress (PSM). These two types are the most common mattresses used in Europe. A Life Cycle Assessment (LCA) shows that the PFM has a higher environmental impact than the PSM. For both products the main cause of environmental impact is the manufacturing process, respectively the polyurethane foam block moulding process for the PFM, and the pocket spring nucleus process for the PSM. A scenario analysis shows the possibility of reducing the environmental impact of the products’ life cycle using an alternative End-of-Life scenario, resorting to incineration rather than landfill. Two strategies were also studied in order to reduce the environmental impact of the PFM: (1) reutilization of foam that was sent to the waste system management, and (2) a 20% weight reduction of the polyurethane foam. The second strategy has proven to be the most effective.