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.

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

Motivation: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. Results: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Amino acids 1-20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES- dependent translation in Hep G2 cells, and inhibit both HCV IRES- and cap-dependent translation in HuH7 and CV-1 cells.

A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1-20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia-HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1-20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.

Computational differentiation of N-terminal signal peptides and transmembrane helices

Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids. This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence. The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology. In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices. Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests. This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy. For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90). Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92). For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83. Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85). The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies. (C) 2003 Elsevier Inc. All rights reserved.

Soybean growth and yield after single tillage and species mixture of cover plants

The use of cover crops is important for the agricultural crop and soil management in order to improve the system and, consequently, to increase yield. Therefore, the present study analyzed the effect of crop residues of black oat (Avena strigosa Schreb.) (BO) and a cocktail (CO) of BO, forage turnip (Raphanus sativus L.) (FT) and common vetch (Vicia sativa L.) (V) on the emergence speed index (ESI), seedling emergence speed (SES) plant height and soybean yield in different intervals between cover crop desiccation with glyphosate 480 (3 L ha-1) and BRS 232 cultivar sowing. Plots of 5 x 2.5 m with 1 m of border received four treatments with BO cover crops and four with CO as well as a control for each cover crop, at random, with five replications. The plots were desiccated in intervals of 1, 10, 20 and 30 days before soybean seeding. The harvest was manual while yield was adjusted to 13% of moisture content. The experimental design was completely randomized with splitplots and means compared by the Scott and Knott test at 5% of significance. The results showed that CO of cover crops can be recommended for soybean to obtain a more vigorous seedling emergence, from 10 days after cover crop desiccation.

Integration of risk management in occupational health and safety systems

Risk management can be considered as part of the Occupational Health and Safety System (OHS) of an organization and can be used to develop and implement the OHS policy and manage the associated risks. The success of the integration of risk management in OHS depends on both technical and human aspects. Thus, this paper presents and discusses the case of a company working in the area of solid waste treatment. This company was certified in 2009 with an Integrated Management Systems for Quality, Environment, Occupational Health and Safety. The evolution of accidents before and after the implementation of the integrated system was analysed and a questionnaire was used to capture the perceptions of the technicians on the risk management system. The analysis of the findings showed that the frequency of accidents increased since 2009 but the severity has been reduced. Several interrelated causes and consequences were analysed and discussed. Furthermore, the analysis of the opinions of the company’s technicians permitted to highlight some important aspects on the integration of risk management in the OHS system of the company. In line with this discussion some hypothesis have been formulated.

Neutron activation analysis of wheat samples

The deficiency of essential micronutrients and excess of toxic metals in cereals, an important food items for human nutrition, can cause public health risk. Therefore, before their consumption and adoption of soil supplementation, concentrations of essential micronutrients and metals in cereals should be monitored. This study collected soil and two varieties of wheat samples–Triticum aestivum L. (Jordão/bread wheat), and Triticum durum L. (Marialva/durum wheat) from Elvas area, Portugal and analyzed concentrations of As, Cr, Co, Fe, K, Na, Rb and Zn using Instrumental Neutron Activation Analysis (INAA) to focus on the risk of adverse public health issues. The low variability and moderate concentrations of metals in soils indicated a lower significant effect of environmental input on metal concentrations in agricultural soils. The Cr and Fe concentrations in soils that ranged from 93–117 and 26,400–31,300 mg/kg, respectively, were relatively high, but Zn concentration was very low (below detection limit <22 mg/kg) indicating that soils should be supplemented with Zn during cultivation. The concentrations of metals in roots and straw of both varieties of wheat decreased in the order of K>Fe>Na>Zn>Cr>Rb>As>Co. Concentrations of As, Co and Cr in root, straw and spike of both varieties were higher than the permissible limits with exception of a few samples. The concentrations of Zn in root, straw and spike were relatively low (4–30 mg/kg) indicating the deficiency of an essential micronutrient Zn in wheat cultivated in Portugal. The elemental transfer from soil to plant decreases with increasing growth of the plant. The concentrations of various metals in different parts of wheat followed the order: Root>Straw>Spike. A few root, straw and spike samples showed enrichment of metals, but the majority of the samples showed no enrichment. Potassium is enriched in all samples of root, straw and spike for both varieties of wheat. Relatively to the seed used for cultivation, Jordão presented higher transfer coefficients than Marialva, in particular for Co, Fe, and Na. The Jordão and Marialva cultivars accumulated not statistically significant different concentrations of different metals. The advantages of using INAA are the multielementality, low detection limits and use of solid samples (no need of digestion).

Validação da eficácia dos procedimentos de limpeza, no equipamento de fabrico e embalagem, na indústria farmacêutica

Na indústria farmacêutica, a limpeza dos equipamentos e superfícies é muito importante no processo de fabrico/embalagem dos produtos farmacêuticos. Possíveis resíduos contaminantes devem ser removidos dos equipamentos e das superfícies envolvidas no processo. De acordo com as Boas Práticas de Fabrico (GMP), os procedimentos de limpeza e os métodos analíticos usados para determinar as quantidades de resíduos devem ser validados. O método analítico combinado com o método de amostragem utilizado na colheita de amostras deve ser sujeito a um ensaio de “recovery”. Neste trabalho apresenta-se uma estratégia inovadora para a validação de limpeza de formas farmacêuticas semi-sólidas. Propõe-se o uso de um método de amostragem que consiste na colheita direta de amostra após o seu fabrico, sendo a análise de resíduos feita directamente nesta amostra. Os produtos escolhidos para a avaliação da estratégia foram dois medicamentos dermatológicos, apresentados na forma de pomada e produzidos numa unidade de fabrico de vários produtos, pela Schering Plough Farma/ Merck Sharp & Dohme (Cacém, Portugal). Como métodos analíticos para a quantificação dos resíduos, utilizaram-se métodos validados por via espectrofotométrica (HPLC), usados na análise do produto acabado. A validação de limpeza foi avaliada através da análise de uma quantidade conhecida de pomada (produto B (*)), usando o método de análise da pomada fabricada anteriormente (produto A (*)), de modo a verificar-se a existência ou não de agente de limpeza e substâncias ativas deixadas após a limpeza do produto A, e vice-versa. As concentrações residuais das substâncias ativas e do agente de limpeza encontradas após a limpeza foram nulas, ou seja, inferiores ao limite de deteção (LOD), sendo que o critério de aceitação da limpeza utilizado foi de 6,4 x 10-4 mg/g para a substância ativa 1 (*); 1,0 x 10-2 mg/g para a substância ativa 2 (*); 1,0 x 10-3 mg/g para a substância ativa 3 (*) e de 10 ppm para o agente de limpeza. No ensaio de “recovery”, obtiveram-se resultados acima de 70% para todas as substâncias ativas e para o agente de limpeza nas duas pomadas. Antes de se proceder a este ensaio de “recovery”, houve a necessidade de ajustar as condições cromatográficas dos métodos analíticos de ambos os produtos e do agente de limpeza, por forma a obter-se valores da conformidade do sistema (fator de tailling e de resolução) de acordo com as especificações. A precisão dos resultados, reportada como desvio padrão relativo (RSD), deu abaixo de 2,0%, excepto nos ensaios que envolvem a substância ativa 3, cuja especificação é inferior a 10,0%. Os resultados obtidos demonstraram que os procedimentos de limpeza usados na unidade de fabrico em causa são eficazes, eliminando assim a existência de contaminação cruzada.

Pesticide residues in Portuguese strawberries grown in 2009–2010 using integrated pest management and organic farming

Pesticides are among the most widely used chemicals in the world. Because of the widespread use of agricultural chemicals in food production, people are exposed to low levels of pesticide residues through their diets. Scientists do not yet have a total understanding of the health effects of these pesticide residues. This work aims to determine differences in terms of pesticide residue content in Portuguese strawberries grown using different agriculture practices. The Quick, Easy, Cheap, Effective, Rugged, and Safe sample preparation method was conducted and shown to have good performance for multiclass pesticides extraction in strawberries. The screening of 25 pesticides residue was performed by gas chromatography–tandem mass spectrometry. In quantitative validation, acceptable performances were achieved with recoveries of 70–120 and <12 % residual standard deviation for 25 pesticides. Good linearity was obtained for all the target compounds, with highly satisfactory repeatability. The limits of detection were in the range of 0.1–28 μg/kg. The method was applied to analyze strawberry samples from organic and integrated pest management (IPM) practices harvested in 2009–2010. The results showed the presence of fludioxonil, bifenthrin, mepanipyrim, tolylfluanid, cyprodinil, tetraconazole, and malathion when using IPM below the maximum residue levels.

Management of agricultural waste on a sample of farms on the island of São Miguel (Açores)

Dissertação de Mestrado em Ambiente, Saúde e Segurança.

A industria da cortiça e o seu potencial de inovação

Este trabalho teve como objectivo a avaliação da actual situação da indústria corticeira, o levantamento de eventuais possibilidades de inovação e o estudo de um caso promissor. Como caso promissor, decidiu-se estudar o efeito da pirólise nos resíduos de cortiça. A análise da situação actual da indústria corticeira aponta para a procura de novos produtos no sentido de alargar o mercado e promover um melhor escoamento deste recurso natural. Apesar de todos os esforços efectuados até ao momento, verifica-se que a indústria vinícola continua a ser o principal mercado da cortiça. O avanço da tecnologia tem permitido o desenvolvimento de novos produtos, alguns ainda em fase de desenvolvimento, e aponta para um potencial de inovação a vários níveis: ao nível do processo de transformação, no sentido da sua optimização; ao nível do desenvolvimento de novos produtos dado o potencial já demonstrado pela cortiça em desenvolvimentos recentes; ao nível da valorização de resíduos como, por exemplo, a consolidação de processo de recuperação de taninos da água de cozedura, a obtenção de suberina e poliois do pó e aparas de cortiça e a pirólise das aparas e pó de cortiça. Como estudo de caso, efectuou-se o estudo do efeito da pirólise em resíduos de cortiça natural, para se poder conhecer as características dos produtos obtidos bem como as condições óptimas de operação. Neste fase inicial e optou-se por se analisar as propriedades parte sólida de modo a saber as alterações sofridas durante a pirólise e estas podem apresentar uma mais-valia para o mercado corticeiro. Para efectuar o respectivo trabalho, recorreu-se a um forno pirolítico horizontal tipo Splitz e utilizou-se cortiça natural com granulometria entre 2,88 e 4,00mm. A pirólise foi realizada entre a gama de temperaturas de 400 e 900ºC e para duas rampas de aquecimento de 5ºC/min e 10ºC/min. O estudo experimental revela que na gama de temperaturas entre os 600 e 800ºC é onde a carbonização do resíduo está completa, sendo para essa mesma gama de temperaturas que se verifica, no resíduo carbonoso, um maior teor de carbono. Relativamente às rampas de aquecimento estas não apresentam efeitos significativos nas massas e teores de carbono no resíduo carbonoso final. Verificouse também, que o teor de hidrogénio diminui com o aumento da temperatura. Conclui-se que a pirólise consegue degradar os resíduos de cortiça levando à libertação de compostos que poderão ser uma mais-valia.

Adding value [valorization] to tannery fleshings: part II – effect of protein hydrolysates on leather dyeing

The tanning industry generates a high quantity of solid wastes. Therefore, there is a need to create valorization [added value] options for these wastes. The main objective of the present work was to study the effect of protein hydrolysates (HP) prepared from fleshings on leather dyeing. During previous studies it was found that the application of HP products, obtained from fleshings, in leather retannage intensified the colour of crust leather. In this work the CIELAB colour system was used to evaluate the effect of HP on retannage processes. The main conclusions of this study were: (i) HP can be used instead of a dicyanodiamide resin (Fortan DC) if the colour parameters of the standard procedure are to be maintained, and (ii) the replacement of an acrylic resin (Fortan A40) by glutaraldehyde-modified HP (GHP) results in a darker skin, and can therefore be interesting for the reduction of the quantity of dye used.

Estudo da produção e aplicação de cola animal

Com as crescentes exigências do mercado e fortes restrições a nível ambiental, as indústrias modernas tendem a progredir no sentido da eficiência, sustentabilidade e rentabilidade dos seus processos produtivos. Sendo a indústria dos curtumes uma forte geradora de resíduos sólidos, é indispensável criar alternativas à valorização desses mesmos resíduos de forma a cumprir todos os requisitos ambientais. Este trabalho remete à reutilização dos resíduos, provenientes da divisão da pele em tripa após operação de descarna, com o intuito de se desenvolver um novo bio-produto apto para ser utilizado noutros sectores industriais. Os resíduos em causa consistem maioritariamente em colagéneo, que apresenta um enorme potencial para se produzir cola animal. Desta forma, este trabalho tem como objectivo principal avaliar a utilização deste tipo de resíduo na produção de um novo bio-produto, cola animal, e testar a sua aplicabilidade técnica e funcional. Com a realização deste trabalho, mostrou-se ser possível produzir um produto, a partir de retalhos da indústria de curtumes, com um perfil de carácter positivo no que diz respeito à viabilidade técnica e funcional da utilização da cola animal como eventual alternativa a um ligante, para formulações de colas de base aquosa para a indústria gráfica e do papel.

Functional domains of Bacillus subtilis transcription factor AraR and identification of aminoacids important for nucleoprotein complex assembly and effector-binding.

Journal of Bacteriology (Apr 2006) 3024-3036