A standard area diagram set for assessing severity of eucalyptus bacterial blight caused by Erwinia psidii / Uma escala diagramática para a avaliação da severidade da seca-de-ponteiros do eucalipto causada por Erwinia psidii

This study aimed to develop and validate a standard area diagram (SAD) set to estimate the severity of bacterial blight of eucalyptus caused by Erwinia psidii. For this purpose, an eight-level SAD was developed and validated by ten inexperienced raters. Accuracy and precision of the estimates by each rater, with and without the SAD, were determined based on Lin's concordance correlation coefficient. The proposed SAD improved the accuracy and precision of the estimates. The SAD set studied here is a useful tool in assessments of bacterial blight of eucalyptus for epidemiological research and breeding programs.(AU)

Este trabalho objetivou o desenvolvimento de uma escala para estimar a severidade da seca-de-ponteiros do eucalipto causada por Erwinia psidii. Para isso, uma escala de oito níveis foi desenvolvida e validada por dez avaliadores inexperientes. A acurácia e precisão das estimativas de cada avaliador, com e sem a escala, foram determinadas baseadas no coeficiente de correlação concordante de Lin. A escala proposta melhorou a acurácia e a precisão das estimativas. A escala estudada se mostrou uma ferramenta útil na avaliação da seca-de-ponteiros do eucalipto para estudos epidemiológicos e em programas de melhoramento.(AU)

L-asparaginase de Erwinia chrysanthemi: expressão proteica livre de células e bioconjugação a bacteriófagos / L-asparaginase from Erwinia chrysanthemi: cell-free protein expression and bioconjugation to bacteriophages

A L-Asparaginase (L-ASNase) de Erwinia chrysathemi (ErA) é uma enzima amplamente utilizada para o tratamento da leucemia linfoblástica aguda (LLA). Embora o seu uso como segunda linha de tratamento para a LLA tenha proporcionado consideráveis benefícios clínicos, reações de hipersensibilidade e rápida depuração plasmática ainda são problemas recorrentes. Ademais, extensivos e custosos processos de produção da ErA são necessários para a obtenção da enzima pura. Com base nesses problemas, o presente trabalho propõe (1) o estudo de viabilidade de expressão da ErA em um sistema de síntese proteica livre de células (SPLC) e (2) a conjugação da proteína em bacteriófagos como ferramenta alternativa para o isolamento e monitoramento da depuração plasmática da ErA. Foram utilizados extratos celulares de Escherichia coli suplementados com solução energética contendo creatina fosfato (CP) como fonte de energia para síntese in vitro de ErA. Para conjugação da ErA a bacteriófagos, o sistema SpyTag/SpyCatcher foi implementado: SpyCatcher foi fusionado à porção N-terminal da ErA e bacteriófagos filamentosos da linhagem M13 e fd foram modificados de modo a expressar SpyTag nas proteínas de capsídeo pIII e pVIII, respectivamente. Em relação ao primeiro objetivo, o sistema de SPLC foi capaz de expressar a ErA com atividade. A proteína foi expressa na fração solúvel e apresentou atividade enzimática significativamente superior em relação à reação controle (7,07 ± 0,68 U/mL vs. 1,83 ± 0,14 U/mL). Tempo necessário para obtenção do extrato celular foi reduzido de 45 para 26 hrs, e sete componentes da solução energética foram removidos da composição original sem implicações negativas na eficiência de expressão da ErA, simplificando desta forma o processo de SPLC. Em relação ao segundo objetivo, ErA fusionada à SpyCatcher (SpyCatcher_ErA) foi conjugada com êxito em bacteriófagos capazes de expressar SpyTag fusionadas na porção N-terminal das proteínas pIII (SpyTag_pIII) e pVIII (SpyTag_pVIII). A porcentagem de formação dos conjugados entre SpyCatcher_ErA e SpyTag_pIII ((ErA)5-pIII) foi de 6% enquanto formação dos conjugados entre SpyCatcher_ErA e SpyTag_pVIII ((ErA)50-pVIII) foi de 46%, valores estes confirmados por atividade enzimática. Solução contendo conjugados foram injetados em camundongos e sequenciados/titulados com êxito. Não houve diferença de depuração plasmática entre (ErA)5-pIII e bacteriófago controle, mas houve maior taxa de eliminação de (ErA)50-pVIII em relação ao mesmo bacteriófago não conjugado à SpyCatcher_ErA. Os resultados aqui apresentados confirmam ser possível expressar ErA com atividade biológica em sistemas de SPLC. Além disso, o sistema de conjugação da ErA a bacteriófagos aqui desenvolvido foi capaz de monitorar a concentração de ErA presente na circulação em função do tempo, tornando-se uma potencial plataforma de desenvolvimento de novas proteoformas da ErA com características clínicas melhoradas

L-Asparaginase (L-ASNase) from Erwinia chrysanthemi (ErA) is a widely used enzyme for treatment of acute lymphoblastic leukemia (ALL). Although its use as a second-line treatment has provided significant clinical benefits, hypersensitivity reactions and a fast clearance rate are recurring L-ASNase-related problems. In addition, extensive and costly production processes are required for the manufacturing of pure ErA. Based on these drawbacks, this current work proposes (1) the study of the use of a cell-free protein synthesis (CFPS) system as a viable platform for the synthesis of ErA and (2) the conjugation of the protein on bacteriophages as an alternative tool for the isolation and monitoring of ErA clearance. Escherichia coli-derived cell extracts supplemented with a creatine phosphate-based energy solution were used to synthesize ErA in vitro. To conjugate ErA on bacteriophages, the SpyTag/SpyCatcher system was implemented: SpyCatcher was fused to the N-terminus of the ErA while filamentous phage strains M13 and fd were engineered in order to display SpyTag on their pIII and pVIII capsid proteins, respectively. Regarding the first goal, the CFPS system was able to express an active ErA. The protein was expressed in the soluble fraction and there presented a significant higher enzymatic activity compared to the control reaction (7.07 ± 0.68 U/mL vs. 1.83 ± 0.14 U/mL). Time required to obtain the cell extract was reduced from 45 to 26 hours, and seven energy solution reagents were removed from the original solution without compromising the efficiency of ErA expression, thus simplifying the CFPS process. With respect to the second goal, ErA fused to SpyCatcher (SpyCatcher_ErA) was sucessfully conjugated on bacteriophages capable of displaying SpyTag fused to the Nterminus of the pIII (SpyTag_pIII) or pVIII (SpyTag_pVIII) proteins. Percentage of conjugate formation between SpyCatcher_ErA and SpyTag_pIII (ErA)5-pIII was 6% whereas conjugate formation between SpyCatcher_ErA and SpyTag_pVIII (ErA)50-pVIII was 46%, values that were confirmed by enzymatic activity. Sample containing conjugates were injected into mice and sucessfully sequenced/titrated. No clearance differences were observed between (ErA)5- pIII and a control bacteriophage, but a higher clearance rate was observed for (ErA)50-pVIII compared to SpyTag_VIII non conjugated to SpyCatcher_ErA. The results here presented confirm the expression of a biologically active ErA from a CFPS system. Besides, the development of a conjugation system capable of linking ErA to bacteriophages could be used as a means to monitor the ErA concentration in the blood as a function of time and also as a potential platform to be used in the development of novel ErA proteoforms with improved clinical properties

Eficacia y seguridad de L-asparaginasa erwinia y L-asparaginasa E. Coli pegilada en el tratamiento de pacientes con leucemia linfoblástica aguda que presentam hipersensibilidad a L-asparaginasa E. coli nativa / Efficacy and safety of L-asparaginase erwinia and L-asparaginase E. coli pegylated in the treatment of patients with acute lymphoblastic leukemia who are hypersensitive to L-asparaginase E. coli native

INTRODUÇÃO: Antecedentes: El presente dictamen expone la evaluación de tecnología de la eficacia y seguridad del uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes con leucemia linfoblástica aguda que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Aspectos Generales: La leucemia es el tipo de cáncer más común en niños, representando aproximadamente el 30% de todos los tipos de cáncer diagnosticados en niños; siendo la leucemia linfoblástica aguda (LLA) uno de los dos tipos de leucemias más comunes. Adicionalmente, alrededor del 60% de todos los casos de LLA ocurre en pacientes menores de 20 años. Así, LLA es un tipo de leucemia de alta importancia dentro de población joven. Tecnología Sanitaria de Interés: Las células neoplásicas en la leucemia linfoblástica aguda (LLA) no sintetizan las cantidades necesarias del aminoácido L-asparagina; por lo que requieren de funtes externas (i.e., L-asparagina extracelular). La L-asparaginasa, es una enzima que cataliza la conversión de L-asparagina más agua, en ácido aspártico y amoniaco, ocasionando que los niveles de L-asparagina extracelular disminuyan; y que por los tanto las células d ela LLA no cuenten con L-asparagina extracelular. Así, estas células neoplásicas se quedan sin fuentes de L-asparagina, y no pueden sintetizar proteínas de gran imporancia para su supervivencia, ocasionando su muerte. METODOLOGÍA: Estrategia de Búsqueda: Se realizó una búsqueda de la literatura a la eficacia y seguridad del uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes niños y adultos con leucemia linfoblástica aguda que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Esta búsqueda se realizó utilizando los meta-buscadores: Translating Research into Practice (TRIPDATABASE) Y National Library of Medicine (Pubmed-Medline). RESULTADOS: Sinopsis de la Evidencia: Se realizó la búsqueda bibliográfica y de evidencia científica hasta marzo del 2017 para el sustento del uso de L-asparaginasa Erwinia en el tratamiento de leucemia linfoblástica aguda en pacientes niños y adultos que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Se presente la evidencia disponible según el tipo de publicación priorizada en los criterios de inclusión (i.e., GP, ETS, RS y ECA fase III), siendo los ensayos de fase III o en su defecto ensayos controlados y aleatorizados la principal considerada. CONCLUSIONES: El presente dictamen evaluó la mejor evidencia disponible hasta marco 2017 en relación al uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensibilidad a L-asparaginasa E. coli nativa. El Instituto de Tecnologías Sanitarias-IETSI, aprueba el uso de L-asparaginasa Erwinia como parte del esquema quimioterápico utilizada para el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensibilidad grado 2 o más a L-asparaginasa E. coli nativa. La vigencia de este dictamen preliminar es de dos años a partir de la fecha de publicación. Asimismo, el Instituto de Tecnologías Sanitarias-IETSI, no aprueba el uso de L-asparaginasa E. coli pegilada en el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensiblidad grado 2 o más a L-asparaginasa E. coli nativa.

Inhibición del crecimiento de Erwinia chrysanthemi a diferentes concentraciones de ácido fólico: posible uso del ácido fólico como agente bacteriostático y fortificante de la papa Solanum tuberosum / Inhibition of Erwinia chrysanthemi growth to different concentrations of folic acid: possible use of folic acid as bacteriostatic agent and fortifying of Solanum tuberosum potato

Resumen Introducción: Las enterobacterias del genero Erwinia spp producen enfermedades en la papa, un tubérculo de consumo masivo. La regulación de la metilación del DNA puede regular la proliferación de la Erwinia, de tal modo que las concentraciones del ácido fólico, pueden tener un efecto en la capacidad patógena del microorganismo. De otra parte, el ácido fólico previene la aparición de defectos del tubo neural en humanos. Objetivo: Evaluar al ácido fólico como un agente bacteriostático de la Erwinia y que a su vez sea parte de la fortificación de alimentos de consumo masivo como la papa. Materiales y métodos: Se llevó a cabo la caracterización bioquímica de la Erwinia chrysanthemi, se estudió su crecimiento frente a diferentes concentraciones de ácido fólico Resultados: Al aumentar las concentraciones de la vitamina, desde 0,3 µg/L hasta 6,8 µg/L se inhibe el crecimiento bacteriano de la Erwinia chrysanthemi. La vitamina inhibe el crecimiento en cultivo de Erwinia chrysanthemi y actúa como como agente bacteriostático, aspecto es de gran relevancia dado que teóricamente, si la papa estuviera fortificada con el micronutriente, este actuaría contra el agente infeccioso y al mismo tiempo contribuiría al consumo adecuado de la vitamina en la población general.

Abstract Introduction: The enterobacteria of the Erwinia spp genus produce disease in potatoes, which is a tuber of mass consumption. The regulation of DNA methylation can regulate the proliferation of Erwinia in such a way that the concentrations of folic acid may have an effect on the microorganism pathogenic ability. On the other hand, the folic acid prevents the appearance of neural tube defects in humans. Objective: To evaluate folic acid as a bacteriostatic agent of Erwinia and, at the same time, as part of the fortification of mass consumption food such as the potatoes. Materials and methods: The biochemical characterization of the Erwinia chrysanthemi was carried out and its growth compared to different concentrations of folic acid was studied. Results: When increasing the concentrations of the vitamin from 0.3 µg/L up to 6.8 µg/L, the bacterial growth of Erwinia chrysanthemi is inhibited. The vitamin inhibits the growth in cultivation of Erwinia chrysanthemi and acts as a bacteriostatic agent. This aspect is of great importance given that, theoretically, if potatoes were fortified with micro-nutrient, this would act against the infectious agent and, at the same time, contribute to the adequate intake of the vitamin in the general population.

Effectiveness of premedication and rapid desensitization in hypersensitivity to L-asparaginase

PURPOSE: L-asparaginase is a crucial chemotherapeutic agent for the treatment of acute lymphoblastic leukemia. However, hypersensitivity to L-asparaginase is common which limits its clinical use. METHODS: We performed 44 cases of premedication and 3 cases of desensitization in 16 patients with hypersensitivity to L-asparaginase. RESULTS: With premedication, 33 cases completed L-asparaginase injection with no hypersensitivity reactions. Eleven cases showed mild hypersensitivity reactions, such as urticaria. Desensitization was performed in 3 cases: in 2 cases, desensitization was successful, and in 1 case the medication was switched to Erwinia asparaginase. CONCLUSION: Premedication and desensitization appear to be useful in helping patients receive desired doses of L-asparaginase in pediatric patients with acute lymphoblastic leukemia.

Lycopene synthesis via tri-cistronic expression of LeGGPS2, LePSY1 and crtI in Escherichia coli / 生物工程学报

Studies on lycopene synthesis in Escherichia coli were not only able to gain the strains with high yield and less by-products, but also able to test functions of genes or gene clusters. In this article, the cDNA sequences of tomato LeGGPS2 and LePSY1 as well as the coding sequence of crtI from Erwinia uredovora, each of which was added a ribosome biding site, were controlled by T7 promoter and terminator alone or combined, and expressed in E. coli BL21 (DE3) to induce lycopene synthesis. The results show that only T7::crtI-LeGGPS2-LePSY1 expressed tri-cistronically could produce lycopene, and 2.124 mg/g dry cell weight oflycopene was obtained when fermented for 5 h at 30 degrees C after mixing 80 micromol/L IPTG at the later logarithmic phase while the seed broth of 1:50 (V/V) was inoculated into LB medium (pH 6.8) containing 3% sucrose and cultured for 8 h at 37 degrees C. The results confirmed the function of the prokaryonized LeGGPS2 and LePSY1 and their synergy with crtI, and also laid a foundation to establish an independent lycopene synthetic pathway in the tomato plastid.

Production of glucosyltransferase by Erwinia sp. using experimental design and response surface methodology

A glicosiltransferase obtida pela linhagem Erwinia sp. é uma enzima intracelular que catalisa a conversão de sacarose em isomaltulose. A isomaltulose é um dissacarídeo redutor, não cariogênico e comercialmente utilizado em alimentos como substituto da sacarose. A metodologia de superfície de resposta e planejamento fatorial composto central-23 foram utilizados para otimizar o meio de cultivo para a producão de glicosiltransferase de Erwinia sp. em frascos sob agitacão a 200 rpm e 30ºC. As três variáveis independentes envolvidas no estudo foram o melaco de cana de acúcar, a água de maceracão de milho e o extrato de levedura Prodex Lac SD. As análises estatísticas dos resultados mostraram que, dentro da faixa estudada das concentracões dos componentes de meio de cultivo, todas as variáveis apresentaram efeito significativo na producão de glicosiltransferase. O meio de cultivo otimizado foi composto de 100 gL-1 de melaco de cana de acúcar, 60 gL-1 de água de maceracão de milho e 8 gL-1 de extrato de levedura Prodex Lac SD, apresentando atividade de glicosiltransferase de 6.65 U mL-1.

Relaciones serológicas entre aislamientos bacterianos de los géneros Erwinia, Pectobacterium Y Pantoea / Relate serológicas among bacterial isolations of the goods Erwinia, Pectobacterium Y Pantoea

Este estudio fue realizado con el propósito de conocer las relaciones serológicas entre aislamientos pertenecientes a diferentes géneros y especies de bacterias. Se utilizaron ocho aislamientos, tres de Pectobacterium Chrysanthemi obtenidos de maíz (Zea mays), papa (Solanum tuberosum) y batata (ipomoea batatas); tres de P. carotovora, provenientes uno de cafecito de jardín (Aglaonema commutatum 'María') y dos de tomate fisiológico y patógeno. Los aislamientos restantes, uno fue obtenido de lechosa (Carica Papaya) y el otro de Alocasia macrorrhiza. Los antisueros se obtuvieron inyectando conejos de 2,5 meses de edad en forma intravenosa con células formalizadas. Los anticuerpos fueron probados con antígenos homólogos y heterólogos como los utilizados en la presente investigación y con otros de los géneros Pseudonomas y Xanthomonas y de contaminantes bacterianos comunes. Los antisueros mostraron buenos títulos y resultaron ser muy específicos. Se establecieron seis serotipos diferentes. El primero (A) conformado por los aislamientos de P. chrysanthemi provenientes de papa y batata y el de carotovora subsp. carotovora de cafecito de jardín. El aislamiento de P. chrysanthemi provenientes de maíz constituyó un serotipo diferente (B). Los dos aislamientos de P. carotovora subsp. carotovora provenientes de tomate, así como los de Erwinia sp. de lechosa y P. aglomerans de Alocasia macrorrhiza, fueron colocadas en diferentes serotipos que se denominaron C, D, E y F respectivamente

A Clinical Study of the Side Effects of L-Asparaginase / 대한소아혈액종양학회지

PURPOSE: We evaluated the frequency and clinical characteristics of the side effects of L-asparaginase during treatment of childrens with acute lymphoblastic leukemia (ALL) & Non-Hodgkin lymphoma (NHL). METHODS: Medical records of children who were treated as 37 ALL and 3 NHL in Korea University Medical Center from January 1995 to June 1998 were reviewed. RESULTS: 1) The 40 patients (21 males and 19 females) had a mean age of 6.2+/-3.3 (range, 0.4~12.8). 2) Nine (7 ALL, 2 NHL) of these 40 patients (median: 5.0 years, range: 3.8~11.5 years) had side reactions (5 hypersensitivity, 2 hyperglycemia, 1 pancreatitis, 1 CNS abnormality) to Escherichia coli L-asparaginase. 3) The hypersensitivity reactions to E. coli asparaginase were the most common side effects. Erwinia asparaginase administered to those patients without side reactions. 4) The range of doses of E. coli asparaginase received prior to experiencing any hypersensitivity was 10 to 22 times. 5) Other reactions (hyperglycemia, pancreatitis and CNS abnormality) were developed at early diseases state and completely recovered. CONCLUSION: We found Erwinia asparaginase to be an acceptable substitute for E. coli asparagianse for childrens who had hypersensitivity reactions. In our experience, hypersensitivity reactions are do not detected in inducton therapeutic period. But, We recommend that you should carefully observe your patients during the early induction period.

Bacterial stem rot of dieffenbachia in Egypt

This work was carried out to isolate and identify the causal organism. Studies included also the host range of the organism. The causal organism was isolated from naturally infected stems of Dieffenbachia picta showing typical rot symptoms. All plants used for isolation were grown in the greenhouse of Faculty of Agriculture in Menia. Four isolates of motile, rod shaped, Gram-negative bacteria were isolated from soft rotted areas. The disease was induced by artificial inoculation of healthy stems of Dieffenbachia with any of these isolates. The bacterial isolates differed in their virulence. Host range studies revealed that the pathogen is able to produce soft rot on fruits or stems of eggplant, tomato, pepper, squash, potato tubers, carrot, turnip and table beet roots. Inoculated stems of chrysanthemum and potato became soft and the softened areas spread rapidly to cause wilting and death of affected plants. Stems of maize and cowpea show no symptoms and remain unaffected. Diagnostic tests and electron microscopic examination suggested that the pathogen as a new species for which the name Erwinia dieffenbachii is proposed. This is the first report on the occurrence of E. dieffenbachii in Egypt

Evaluation of different Bradyrhizobium sp strains on nityrogen fixation capacity in two forage legumes

Nineteen avaiable strains of Bradyrhizobium sp were used to test two forage legumes, Crotalaria juncea(Crotalaria) and Cajanus cajans (Guandu) for symbiotic N-fixation ability. Greenhouse experiments were conducted for nine weeks in order to evaluate shoot, root, and nodule dry weight, nodule number, and shoot nitrogen content. Nine strains presented relative nitrogen efficiency (RNE) over 100 per cent with Crotalaria cultivar, whereas only 3 strains were more efficient with Guandu. Strain 633 was the only which presented a high yield with both cultivars. Finally, plant dry weight accumulation followed N accumulation obtaining 0.6757 correlation coefficient for Crotalaria and Guandu, respectively

Purification and preliminary characterization of L-asparaginase from Erwinia aroideae NRRL B-138.

L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) from Erwinia aroideae NRRL B-138 has been purified to apparent homogeneity by ammonium sulphate precipitation, chromatography on sulfopropyl-sephadex C-50 and sephadex G-200 with 22% recovery and 567-fold purification. The enzyme obtained from sulfopropyl-sephadex C-50 was unstable and lost activity within a few hours. Addition of glycerol helped in restoring the activity of the enzyme. The enzyme has an apparent molecular mass of approximately 155 kDa and has four subunits of identical molecular mass of approximately 38 kDa. The K(m) for L-asparagine is 2.8 x 10(-3) M. Enzyme shows optimal activity at 45 degrees C and pH 8.2. Energy of activation as determined from Arrhenius plot was 9.1 kcal/mol. Substrate L-asparagine and analogue L-glutamine, D-asparagine and 6 diazo-5-oxo-L-norleucine provide full protection to the enzyme against thermal denaturation.

Studies of the antifungal compounds produced by Erwinia herbicola.

The organic phase of a wide spectrum, antimycotic and diffusable toxin from Erwinia herbicola showed a highly significant inhibitory activity against Pyricularia oryzae spores in spore well bioassay. Germ tube lengths were inhibited more in wells containing 5 microliters equivalent of bacterial toxin than 1 microliter. No significant difference between the germ tube in an equal mixture of Dimethyl sulphoxide: ethanol and controls. Thin layer chromatography using the chloroform extraction of the organic phase showed a significant antagonism on Cladosporium cucumerinum. The retardation factor values for inhibitory zones in solvent 1 were 0.07 for lower spot and 0.26 for upper spot.

Purification and characterization of pectin lyase of erwinia dissolvent

Erwinia dissolvens [Code No. E2 F2] was grown in two different media by static culture method for two weeks. The extracellular pectin lyase produced by the organism in Medium I was partially purified by gel filtration chromatography on Sephadex G-75 and Sephadex G-200 while that produced in Medium II was purified by chromatographing on Sephadex G-75, Sephadex G-200, DEAE-cellulose columns. Some characteristics of the enzyme were determined and compared with those of pectin lyases and pectate lyases of bacterial and fungal origins previously reported in the literature