Inhibición de enzimas fosfolipasas A2 de venenos totales de serpientes como estrategia en la búsqueda de nuevos fármacos / Enzyme phospholipase A2 inhibition from snake venoms as strategy to search for new drug

Introducción: Los fármacos antiinflamatorios no esteroideos (AINEs) son ampliamente utilizados para la terapia del dolor, a pesar de sus efectos secundarios que ocurren a nivel renal, estomacal y coagulatorio. Las fosfolipasas A2 (PLA2) presentes en los venenos de serpientes, abejas e incluso en el organismo humano, son responsables de varios procesos fisiológicos y patológicos. Las enzimas hidrolizan fosfolípidos de membrana liberando ácido araquidónico, un precursor de los eicosanoides pro-inflamatorios, los cuales originan mediadores de la inflamación. Objetivo: El propósito de este trabajo es revisar nuevas moléculas capaces de bloquear la escisión de los fosfolípidos de membrana por acción de las PLA2, evitando la formación de mediadores de inflamación. Metodología: Se realizó una revisión bibliográfica de estudios publicados desde 2011 a 2021, que reportan compuestos con actividad inhibitoria frente a PLA2. El potencial de los estudios de relación estructura actividad se discute como estrategia para encontrar compuestos activos ante PLA2. Resultados: Se revisaron 26 estudios que incluyen compuestos naturales y sintéticos y se recopilaron 93 moléculas con actividad inhibitoria, destacando su potencial como inhibidores de PLA2. Conclusiones: La actividad inhibitoria de los compuestos revisados podría estar asociada a los patrones de sustitución en el anillo bencénico de las moléculas. La evaluación de características moleculares relevantes en la inhibición de PLA2 puede guiar a la identificación de candidatos para síntesis de nuevos inhibidores enzimáticos.

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for pain therapy, despite its side effects in renal, stomach and coagulant systems. Phospholipases A2 (PLA2) enzymes, present in snakes and bees' venoms, and even in the human organism, are responsible for several physiological and pathological processes. These enzymes hydrolyze membrane phospholipids, releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids, which give rise to inflammatory mediators. Objective: The aim of the present work is to review new molecules able to block the cleavage of membrane phospholipids by the action of phospholipases A2, preventing the formation of inflammatory mediators. Methodology: A bibliographic revision from literature published from 2011 to 2021 focused on PLA2 inhibitors was carried out. The potential of structure-activity relationship studies is discussed as a strategy to find active compounds against PLA2. Results: 26 studies including natural and synthetic compounds were reviewed and data from 93 molecules with inhibitory activity were collected, highlighting its potential as PLA2 inhibitors. Conclusion: The inhibitory activity of the reviewed compounds could be associated with the substitution patterns in the benzene ring of the molecules. The evaluation of molecular moieties with relevant capacity to inhibit PLA2 will lead to the identification of candidates for synthesis of new enzymes inhibitors.

Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying / 生物工程学报

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.

Effect of Inoculation with Organophosphate Solubilizing Bacteria on Quality and Soil Fertility of Paris polyphylla var. yunnanensis / 中国实验方剂学杂志

ObjectiveThe effect of inoculation with different organophosphate-resolving bacteria or compound bacteria on the quality of Paris polyphylla var. yunnanensis medicinal materials and rhizosphere soil fertility were studied to provide a reference for the development and application of biological bacterial fertilizer in artificial cultivation of P. polyphylla var. yunnanensis. MethodThe three dominant species of organophosphate-solubilizing bacteria were inoculated separately and in combination in sterilized soil by single-factor indoor pot planting， and no inoculation was used as the control group. The effect of inoculation of organophosphate-solubilizing bacteria on total saponins content in rhizomes of P. polyphylla var. yunnanensis， as well as microbial numbers， enzyme activities and nutrient contents in rhizosphere soil were analyzed. ResultIn the seven treatments inoculated with organophosphate-solubilizing bacteria， the total saponin content in the rhizomes of Paris polyphylla var. yunnanensis was increased by 16.42%， 3.83%， 16.86%， 33.69%， 2.11%， 13.44%， and 28.83%， respectively， compared with the control. Inoculation with organophosphate-solubilizing bacteria increased the number of soil microorganisms to varying degrees， and the effects of S6 and S7 treatments were the most significant. Inoculation with organophosphate-solubilizing bacteria improved the enzyme activity， and the effect of S7 treatment was the most significant. The activities of acid phosphatase， neutral phosphatase， alkaline phosphatase， protease， invertase and catalase were 49.96% and 104.67% ， 110.17%， 99.61%， 26.26%， 11.29% higher than those of the control， respectively. Inoculation with organophosphate-solubilizing bacteria reduced the pH of the rhizosphere soil and increased the content of soil available nutrients. Under the S7 treatment， the contents of alkaline hydrolyzable nitrogen， available phosphorus and available potassium in the rhizosphere soil of P. polyphylla var. yunnanensis were 181.46%， 51.64% and 42.62% higher than those of the control， respectively. Correlation analysis showed that there was a significant positive correlation between total saponins and phosphatase activities， a significant positive correlation between soil microorganisms and soil enzyme activities， and a very significant positive correlation between soil nutrients. ConclusionInoculation of different organophosphate-resolving bacteria or compound bacteria can improve the quality and rhizosphere soil fertility of P. polyphylla var. yunnanensis. Among them， the mixed inoculation of three kinds of bacteria and the mixed inoculation of B. mycoides and B. wiedmannii have the best effect.

Metabolitos secundarios aislados de especies de la familia Myristicaceae que producen inhibición enzimática / Secondary metabolites of species of the Myristicaceae family that produce enzymatic inhibition / Metabólitos secundários isolados de espécies da família Myristicaceae que produzem inibição enzimática

RESUMEN Introducción: Diferentes especies de la familia Myristicaceae han sido utilizadas con fines medicinales, nutricionales e industriales, mostrando así la importancia y potencial de la familia en diversos campos. El uso medicinal ha sido primordial por diferentes comunidades indígenas y ha venido en aumento como alternativa al tratamiento de enfermedades, por lo cual la investigación se está concentrando en la medicina herbaria y plantas medicinales, siendo este el primer paso para el desarrollo e innovación de fármacos. Entre los tipos de fármacos se destacan los inhibidores enzimáticos, que actúan regulando procesos metabólicos o atacando patógenos. Objetivos: Recopilar información de la herboristería de la familia Myristicaceae que incluya aspectos de fitoquímica, etnobotánica, usos industriales, actividad biológica y determinar posibles metabolitos secundarios que producen inhibición enzimática y actividad biológica. Metodología: La búsqueda de información se realizó con artículos científicos, libros especializados y tesis de grado. Resultados y conclusiones: Se encontró que compuestos fenólicos de tipo: acilfenol, lignano, neolignano, flavonoide, alquenil-fenol, tocotrienol y ácido fenólico producen inhibición enzimática. Los compuestos fenólicos identificados en especies de la familia Myristicaceae son una fuente promisoria de metabolitos que producen inhibición enzimática. Siendo los metabolitos de tipo lignanos los que presentaron mayor número de estudios de inhibición enzimática. La información analizada puede servir de base para el desarrollo de investigaciones de relación estructura actividad y/o acoplamiento molecular entre metabolitos secundarios y enzimas inhibidas, con especies de la familia Myristicaceae.

SUMMARY Introduction: Different species of the Myristicaceae family have been used for medicinal, nutritional and industrial purposes, showing the importance and potential of the family in various fields. Medicinal use has been primordial for different indigenous communities and has been increasing as an alternative for the treatment of diseases, for which research is concentrating on herbal medicine and medicinal plants, being this the first step for the development and innovation of pharmaceuticals. Among the types of drugs, enzymatic inhibitors, which act by regulating metabolic processes or attacking pathogens, stand out. Objectives: To gather information on the herbalism of the Myristicaceae family including aspects of phytochemistry, ethnobotany, industrial uses, biological activity and to determine possible secondary metabolites that produce enzymatic inhibition and biological activity. Methodology: The search for information was carried out using scientific articles, specialized books and degree theses. Results and conclusions: Phenolic compounds of type: acylphenol, lignan, neolignan, flavonoid, alkenylphenol, tocotrienol and phenolic acid were found to produce enzymatic inhibition. Phenolic compounds identified in species of the Myristicaceae family are a promising source of metabolites that produce enzymatic inhibition. Lignan-type metabolites were the ones that presented the greatest number of enzymatic inhibition studies. The information analyzed can serve as a basis for the development of research on the structure-activity relationship andlor molecular coupling between secondary metabolites and inhibited enzymes, with species of the Myristicaceae family.

RESUMO Introdução: Diferentes espécies da família Myristicaceae têm sido utilizadas para fins medicinais, nutricionais e industriais, mostrando a importância e o potencial da família em vários campos. O uso medicinal tem sido de suma importância para diferentes comunidades indígenas e tem aumentado como alternativa para o tratamento de doenças, razão pela qual a pesquisa está se concentrando na medicina herbal e plantas medicinais, sendo este o primeiro passo para o desenvolvimento e inovação de produtos farmacêuticos. Entre os tipos de drogas estão os inibidores enzimáticos, que atuam regulando processos metabólicos ou atacando patógenos. Objetivos: Compilar informações sobre o herbalismo da família Myristicaceae, incluindo aspectos de fitoquímica, etnobotànica, usos industriais, atividade biológica e determinar possíveis metabolitos secundários que produzem inibição enzimática e atividade biológica. Metodologia: A busca de informações foi realizada utilizando artigos científicos, livros especializados e teses de graduação. Resultados e conclusões: Foram encontrados compostos fenólicos como: acylphenol, lignan, neolignan, flavonoid, alkenylphenol, tocotrienol e ácido fenólico para produzir inibição enzimática. Os compostos fenólicos identificados em espécies da família Myristicaceae são uma fonte promissora de meta-bólitos que produzem inibição enzimática. Os metabólitos do tipo lignano mostraram o maior número de estudos de inibição enzimática. As informações analisadas podem servir como base para o desenvolvimento de pesquisas sobre a relação estrutura-ativi-dade eIou acoplamento molecular entre metabolitos secundários e enzimas inibidas, com espécies da família Myristicaceae.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Estudo da estabilidade e peguilação de L-asparaginase em soluções aquosas de líquidos iônicos de imidazólio / Study of the stability and pegylation of L-asparaginase in aqueous imidazole ionic liquid solutions

A enzima L-asparaginase de Escherichia coli (ASNase) é um biofármaco indicado para o tratamento de leucemia linfoblástica aguda, mas que pode causar reações de hipersensibilidade nos pacientes tratados. Na tentativa de amenizar esse efeito, foi desenvolvida a PEG-ASNase (enzima conjugada com polietilenoglicol) que apresenta a vantagem de ser menos imunogênica e ter maior meia-vida biológica. Mais recentemente, novas abordagens têm sido desenvolvidas visando aprimorar os processos de PEGuilação por meio de reações sítio dirigidas, por exemplo N-terminal, a fim de promover maior similaridade lote a lote e controle das características farmacocinéticas e farmacodinâmicas do biofármaco. Porém, existe ainda uma limitação associada à hidrólise do PEG reativo, desta forma surge a necessidade de procurar solventes alternativos para a PEGuilação que permitam manter a estabilidade das proteínas, aumentar o rendimento de PEGuilação e a estabilidade do PEG reativo. Nesse trabalho, líquidos iônicos foram investigados como solventes alternativos para a peguilação N-terminal de PEG-ASNase. Para tal, a estabilidade de ASNase em Lis foi investigada em LIs da família metil-imidazol, analisando a influência do aumento da cadeia alquílica e de diferentes ânions. A estabilidade da ASNase é favorecida quando em contato com Lis relativamente hidrofóbicos ([C2mim]Cl, [C4mim]Cl e [C6mim]Cl), mas sua a atividade é prejudicada quando o LI é muito polar, como o [C4mim][(CH3)2PO4] ou anfifílico como o [C12mim]Cl. Apesar de seu efeito desnaturante, o [C4mim][(CH3)2PO4] resultou no maior rendimento da reação de PEGuilação da ASNase (56%) quando empregado a 75% e a reação realizada em 10 min. O [C4mim]Cl resultou em rendimento semelhante ao tampão fosfato (~ 49%), mas ambos os LIs reduziram a poliPEGuilação. Portanto, os Lis [C4mim]Cl e [C4mim][(CH3)2PO4] fornecem uma alternativa viável à reação de PEGuilação pela redução na formação de espécies poliPEGuiladas, o que facilitaria os processos de purificação e permitiria maior controle lote a lote da reação, bem como pelo aumento do rendimento da reação no caso do [C4mim][(CH3)2PO4]

Escherichia coli L-asparaginase enzyme (ASNase) is a biopharmaceutical indicated for the treatment of acute lymphoblastic leukemia, but may cause hypersensitivity in the patients used. In an attempt to alleviate this effect, PEG-ASNase (polyethylene glycol conjugated enzyme) was developed, which has the advantage of being less immunogenic and having a longer biological half-life. More recently, new approaches have been applied to improve PEGylation processes through targeted sites, for example N-terminal, in order to promote greater similarity to the batch and control of the pharmacokinetic and pharmacodynamic characteristics of the biopharmaceutical. However, there is still a limitation associated with reactive PEG hydrolysis, thus increasing the need to look for alternative PEGylation solvents to maintain protein stability, increase PEGylation yield and use reactive PEG. In this work, ions were investigated as alternative solvents for the N-terminal PEG-ASNase. For example, a stability of ASNase in ILs was investigated in imidazole ILs by analyzing the influence of increased alkyl chain and different anions. ASNase stability is enhanced when in contact with relatively hydrophobic ILs ([C2min]Cl, [C4min]Cl and [C6min]Cl), but its activity is impaired when very polar ILs such as [C4min][(CH3)2PO4] or amphiphilic as [C12mim]Cl. Despite its denaturing effect, [C4min][(CH3)2PO4] resulted in higher yield of ASNase PEGylation reaction (56%) when employed at 75% and reaction performed in 10 min. [C4min]Cl yielded similar phosphate buffer yield (~ 49%), but both ILs reduced polyPEGylation. Therefore, [C4min]Cl and [C4min][(CH3)2PO4] Ils may use a viable alternative to the PEGylation reaction and reduce the formation of polyPEGylated species, or that facilitate purification processes and allow for greater batch use of the solution, as well as increased reaction yield in the case of [C4min][(CH3)2PO4]

Enzymatic properties of α-L-rhamnosidase and the factors affecting its activity: a review / 生物工程学报

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.

Analytical methodologies for sensing catechol-O-methyltransferase activity and their applications / 药物分析学报

Mammalian catechol-O-methyltransferases(COMT)are an important class of conjugative enzymes,which play a key role in the metabolism and inactivation of catechol neurotransmitters,catechol es-trogens and a wide range of endobiotics and xenobiotics that bear the catechol group.Currently,COMT inhibitors are used in combination with levodopa for the treatment of Parkinson's disease in clinical practice.The crucial role of COMT in human health has raised great interest in the development of more practical assays for highly selective and sensitive detection of COMT activity in real samples,as well as for rapid screening and characterization of COMT inhibitors as drug candidates.This review summarizes recent advances in analytical methodologies for sensing COMT activity and their applications.Several lists of biochemical assays for measuring COMT activity,including the probe substrates,along with their analytical conditions and kinetic parameters,are presented.Finally,the challenges and future perspec-tives in the field,such as visualization of COMT activity in vivo and in situ,are highlighted.Collectively,this review article overviews the practical assays for measuring COMT activities in complex biological samples,which will strongly facilitate the investigations on the relevance of COMT to human diseases and promote the discovery of COMT inhibitors via high-throughput screening.

Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.

How different soil moisture levels affect the microbial activity / Como diferentes níveis de umidade do solo afetam a atividade microbiana de um solo de terras baixas usado para cultivo do arroz?

ABSTRACT: The lowland soils are characterized by high susceptibility to water saturation. This anaerobic condition is usually reported in paddy fields and alters the decomposition process of soil organic compounds. The aim of this study was to evaluate the soil microbial and enzymatic activity of a lowland soil at different soil moisture contents. A poorly drained Albaqualf cultivated with irrigated rice was used to evaluate microbial and enzymatic activity in treatments with different levels of soil moisture, being: i) 60% of field capacity (FC) (60%FC); ii) 100% of FC (100%FC); iii) flooded soil with a 2 cm water layer above soil surface, and iv) soil kept at 60%FC with late flood after 29 days the incubation. The greater soil microbial activity was observed in the 100%FC treatment, being 41% greater than 60%FC treatment and only 2% higher than flooded treatment. The enzymatic activity data by fluorescein diacetate (FDA) hydrolysis corroborated the higher CO2 release in treatments with higher soil moisture content. Differently from the results reported, the main methodologies to evaluate microbial activity still recommend maintenance of the soil with a moisture content close to 60% of the FC. However, in lowland soil with history of frequent paddy fields, the maintenance of moisture close to 60% of the FC can limit the microbial activity. The soil respiration technique can be used to evaluate the microbial activity in flooded soil conditions. However, further studies should be conducted to understand the effect of the cultivation history on the microbial community of these environments.

RESUMO: Os solos de várzea são caracterizados pela alta suscetibilidade à saturação por água. Esta condição anaeróbica é geralmente encontrada em solos arrozeiros e altera o processo de decomposição dos compostos orgânicos do solo. O objetivo deste estudo foi avaliar a atividade microbiana e enzimática de um solo de várzea sob diferentes teores de umidade no solo. Um Planossolo historicamente cultivado com arroz irrigado foi utilizado para avaliar a atividade microbiana e enzimática em tratamentos com diferentes níveis de umidade do solo, sendo: i) 60% de capacidade de campo (CC) (60%CC); ii) 100% da CC (100%CC); iii) solo inundado com uma camada de água de 2 cm acima da superfície do solo; e iv) solo mantido a 60%CC com inundação após 29 dias da incubação. A maior atividade microbiana do solo foi observada no tratamento 100%CC, sendo 41% maior que o tratamento 60%CC e 2% maior que tratamento inundado. Os dados da atividade enzimática pela hidrólise do diacetato de fluoresceína (FDA) corroboraram a maior liberação de CO2 nos tratamentos com maior umidade do solo. Diferentemente dos resultados encontrados, as principais metodologias para avaliação da atividade microbiana ainda recomendam manter o solo com umidade próxima a 60% da CC. No entanto, em solos de várzea com históricos de cultivos de arroz, a manutenção de umidade próxima a 60% da CC pode limitar a atividade microbiana. A técnica de respiração do solo pode ser usada para avaliar a atividade microbiana em condições de solo inundado. No entanto, mais estudos devem ser realizados para entender o efeito do histórico de cultivo na comunidade microbiana nesses ambientes.

Efecto de la procedencia y el almacenamiento en la calidad del café / Effect of provenance and storage on coffee quality

Resumen Antecedentes. La calidad del grano de café ha sido relacionada con su procedencia, su manejo agronómico y sus condiciones de almacenamiento. Objetivo. Determinar la actividad de la polifenil oxidasa, el contenido de lípidos, el color y las características organolépticas de cafés provenientes de 3 subestaciones experimentales. Materiales y métodos. Se siguió un diseño completamente aleatorio en arreglo factorial factorial 3x6 (lugares de procedencia del café y tiempo de almacenamiento respectivamente). Resultados. La actividad de la polifenil oxidasa es mayor en el café fresco-para las tres procedencias. El café procedente de Naranjal presentó actividades enzimáticas más altas que los cafés provenientes de las subestaciones Supía y la Catalina. El análisis de varianza mostró el efecto de la procedencia sobre la variable actividad enzimática. La actividad de la polifenil oxidasa en los cafés estudiados decrece con el tiempo de almacenamiento. El contenido de lípidos es menor a menor en la subestación de la Catalina. Todos los cafés fueron caracterizados de buena calidad en el tiempo cero de almacenamiento. Las características de aroma, intensidad del aroma y cuerpo presentaron altibajos en los diferentes meses de almacenamiento. El café de Naranjal, obtuvo en promedio una calificación aceptable a lo largo de los seis meses de almacenamiento. Conclusiones. Se encontraron diferencias significativas para las variables estudiadas por efecto de la procedencia y el almacenamiento. La actividad enzimática de la PFO presentó etapas de activación/inhibición, durante los seis meses de almacenamiento.

Abstract Background. The quality of the coffee bean has been related to its origin, the agronomic management and the storage conditions. Objective. To determine the activity of the polyphenyl oxidase, the lipid content, the color and the organoleptic characteristics of coffees from 3 experimental substations. Materials and methods. A completely randomized design was followed in a 3x6 factorial arrangement (places of coffee origin and storage time respectively). Results. The activity of polyphenyl oxidase is greater in fresh coffee-for the three provenances. The coffee from Naranjal presented higher enzymatic activities than the coffees from the Supía and the Catalina substations. The analysis of variance showed the effect of provenance on the enzyme activity variable. The activity of the polyphenyl oxidase in the coffees studied decreases with storage time. The lipid content is lower at a lower height in the Catalina. All coffees were characterized as good quality at zero storage time; but the characteristics of aroma, intensity of aroma and body presented ups and downs in the different months of storage. Naranjal coffee, on average, obtained an acceptable rating throughout the six months of storage. Conclusions. Significant differences were found for the variables studied due to the effect of provenance and storage. The enzymatic activity of the PFO showed activation / inhibition stages, during the six months of storage.

Método microespectrofotométrico para la determinación de valores de referencia de la fosfomanosa isomerasa / A Microspectrophotometric Method for the Determination of Reference Values of Phosphomannose Isomerase / Método microespectrofotométrico para determinar valores de referência da fosfomanose isomerase

Resumen: Los desórdenes congénitos de glicosilación son un conjunto de defectos genéticos de tipo multisistémico que afectan la función de la proteína. Se han descrito cerca de 75 enfermedades desde sus primeros estudios. En el presente estudio se desarrolló un método microespectrofotométrico para el diagnóstico de la enzima citosólica fosfomanosa isomerasa EC 5.3.1.8 (PMI), se analizaron 32 muestras de individuos con rango de edad de 0,6 a 27 años y se estableció el intervalo y el valor de referencia de actividad enzimática específica. Este estudio permitirá iniciar el diagnóstico de pacientes deficientes de la PMI de forma temprana y oportuna, lo cual la convierte en una posible enzima candidata para pruebas de tamizaje neonatal, ya que esta patología tiene un tratamiento fácil y de bajo costo, que consiste en la suplementación de manosa en forma oral. El diagnóstico clínico de este desorden metabólico beneficiará al paciente y a su familia al mejorar su calidad de vida, como también al sistema de salud colombiano.

Abstract: Congenital glycosylation disorders are a set of multi-systemic genetic defects affecting protein function. About 75 diseases have been described since early studies. This study developed a microspectrophotometric method for the diagnosis of the cytosolic enzyme phosphomannose isomerase (PMI) (EC 5.3.1.8), analyzed 32 samples of individuals ranging between 0.6 and 27 years old, and established the interval and reference value of specific enzyme activity. This study will allow early and timely diagnosis of PMI deficient patients, which makes this enzyme a potential candidate for neonatal screening tests since this pathology has an easy, low-cost treatment (oral administration of mannose supplements). Clinical diagnosis of this metabolic disorder will benefit the patient and his family by improving his quality of life, as well as the Colombian healthcare system.

Resumo: Os defeitos congénitos de glicosilação são um conjunto de defeitos genéticos de tipo mul-tissistêmico que afetam a função da proteína. Foram descritas 75 doenças desde seus primeiros estudos. Neste estudo, foi desenvolvido um método microespectrofotométrico para diagnosticar a enzima citosólica fosfomanose isomerase EC 5.3.1.8 (PMI); foram analisadas 32 amostras de indivíduos entre 0,6 e 27 anos e estabelecidos o intervalo e o valor de referência de atividade enzimática específica. Este estudo permitirá iniciar o diagnóstico de pacientes deficientes da PMI de forma precoce e oportuna, o que a converte em uma possível enzima candidata para testes genéticos de rastreio pré-natal, já que essa patologia tem um tratamento fácil e de baixo custo, que consiste na suplementação de manose por via oral. O diagnóstico clínico desse defeito metabólico beneficiará o paciente e sua família ao melhorar a qualidade de vida e o sistema de saúde colombiano.

Effects of bovine enterovirus and type 1 diabetes on liver and kidney pyruvate kinase activity in an animal model

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic beta cells. In addition to genetic factors, enteroviruses have been considered the main environmental factor involved in this pathology. Therefore, the objective of this study was to evaluate the effects of streptozotocin-induced diabetes and bovine enterovirus (BEV) on liver and kidney pyruvate kinase activity in rats. Fourteen male Wistar rats were divided in three groups: control, diabetes and a third group, which was fed with water experimentally contaminated by BEV. Increased blood glucose levels were found in both diabetes and enterovirus groups, whereas there were no alterations in the lipid profile. A reduced pyruvate kinase activity was observed in the liver and kidney of animals from diabetes and enterovirus groups. Under our experimental conditions, the ingestion of water experimentally contaminated by BEV induced alterations in glycaemia, and also interfered in the pyruvate kinase activity in liver and kidney of the rats, which might be one of the possible mechanisms involved in the T1D development.

Esterase activity in homogenates of Helicoverpa armigera (Hubner) (Lepidoptera: noctuidae) exposed to different insecticides and the behavioral effect / Atividade esterase em homogenatos de helicoverpa armigera (hubner) (lepidoptera: noctuidae) expostos a diferentes inseticidas e o efeito comportamental

The toxicity of some insecticides to Helicoverpa armigera was studied through sublethal effects, evaluating the enzymatic activity of esterase and the behavioral response. The commercial formulation of insecticides selected were chlorpyrifos, spinosad, indoxacarb, chlorantraniliprole, lambda-cyhalothrinand Bacillus thuringiensis, corresponding the most used by farmers to control of H. armigera. To determine the esterase activity, the larvae were fed with soybean leaf discs dipped in insecticide solution using the lethal concentration (LC50). For the behavioral response, filter paper were immersed in three concentrations of insecticides (LC50, LC95 and recommend dose.), then the behavior of the larvae observed in Videomex-One. The results for the enzymatic activity showed an increase in the activity of the esterase, with variation along the treatments and the time of exposure of the insects to the insecticides. With exception of spinosad, other insecticides showed an increase in EST-α activity, 6 and 24 hours after contact of caterpillars with insecticide.Different behavioral patterns of walking (walking distance, walking speed and resting time) were observed for H. armigera exposed to different insecticides. H. armigera exposed to chlorpyrifos, lambda-cyhalothrin and B. thuringiensis insecticides show greater esterase activity. Regarding walking behavior, the results confirms enzymatic activity, where H. armigera have behavioral alteration after exposure to insecticide.

A toxicidade de alguns inseticidas a Helicoverpa armigera foi estudada através de efeitos subletais, avaliando a atividade enzimática da esterase e a resposta comportamental. A formulação comercial dos inseticidas selecionados foram clorpirifos, espinosad, indoxacarb, clorantraniliprole, lambda-cialotrina eBacillus thuringiensis utilizados pelos agricultores para controle de H. armigera. Para determinar a atividade da esterase, as larvas foram alimentadas com discos de folha de soja mergulhados em solução de inseticida usando a concentração letal (CL50). Para os testes de características comportamentais, papel filtro foi imerso em três concentrações de inseticidas (CL50, CL95 e dose recomendada), posteriormente o comportamento das larvas foi observado em Videomex-One. Os resultados para a atividade enzimática mostraram aumento da atividade daesterase, com variação ao longo dos tratamentos e do tempo de exposição dos insetos aos inseticidas. Com exceção do spinosad, outros inseticidas mostraram um aumento na atividade EST-α, 6 e 24 horas após o contato das lagartas com inseticida. Diferentes padrões comportamentais de caminhada (distância percorrida, velocidade de caminhada e tempo de repouso) para H. armigera exposto a diferentes classes de inseticidas. H. armigera exposta aos inseticidas clorpirifos, lambda-cialotrina e B. thuringiensis mostraram maior atividade da esterase. Em relação ao comportamento ambulante, os resultados confirmam a atividade enzimática, onde H. armigera teve alteração comportamental após exposição ao inseticida.

Inhibitory effects of down-regulation of JNK enzymatic activity on growth of human prostate cancer cells, hepatocellular carcinoma cells and breast cancer cells / 吉林大学学报(医学版)

Objective: To discuss the role for down-regulation of JNK enzymatic activity in the tumor treatments by interrupting JNK gene expression with JNK inhibitor SP600125 and JNK-siRNA encapsulated by lipid nanoparticles in vivo and in vitro. Methods: In vitro experiments, the experiment was divided into siRNA group and inhibitor SP600125 group. In siRNA group, JNK-siRNA and NC-siRNA were transfected into the human prostate cancer cells (PC cells), human hepatocellular carcinoma cells (SMMC-7721 cells) and human breast cancer cells (MCF cells), respectively. In inhibitor SP600125 group, SP600125 was delivered to human hepatocellular carcinoma cells. The expression levels of JNK or p-JNK proteins in human prostate cancer cells, human hepatocellular carcinoma cells, and human breast cancer cells after transfected with JNK-siRNA and inhibitor SP600125 were detected by Western blotting method. The cell viabilities of tumor cells in various groups were examined by WST-1 proliferation assay. In vivo experiments, the human hepatocellular carcinoma SMMC-7721 cells were used to establish the subcutaneous hepatocellular carcinoma xenograft tumor models by subcutaneous injection. The eight mice were fed until the tumors were generated to 3 mm× 3 mm. They were randomly divided into inhibitor SP600125 group and negative control group, JNK-siRNA group and NC-siRNA control group. Each mouse in various groups was injected intratumorally with 5 nmol SP600125, negative control solution, lipid nanoparticles-mediated JNK-siRNA and NC-siRNA negative control. The volumes of tumors of the mice in various groups were observed. The expressions of JNK or p-JNK proteins in tumor tissue were examined by immunohistochemical staining. Results: In vitro experiments, compared with NC-siRNA control group, the expression level of JNK protein in human prostate cancer cells, human hepatocellular carcinoma cells and human breast cancer cells in JNK-siRNA group were decreased (P<0. 01); the expression level of p-JNK protein in human hepatocellular carcinoma cells in inhibitor SP600125 group was significantly increased compared with its negative control group (P<0. 01). Invivo experiment, the human hepatocellular carcinoma cells were taken as an example, the volume of tumor of the mice in inhibitor SP600125 group was significantly reduced compared with negative control group, whereas the change of tumor volume in JNK-siRNAs group was not significant compared with NC-siRNA control group. The immunohistochemical staining results showed that compared with their negative control groups, the expression amount of p-JNK protein in tumor tissue of the mice in SP600125 group and the expression amount of JNK protein in tumor tissue of the mice in JNK-siRNA group were decreased (P< 0.01). Conclusion: Down-regulation of enzymatic activity of JNK can decrease the expression level of JNK gene and then inhibit the tumor growth both in vivo and in vitro.

PAF-AH and PLA2 activity of Leptospira interrogans LA_2144 gene product / 中华微生物学和免疫学杂志

Objective To analyze the enzymatic activity of Leptospira interrogans ( L. interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase ( PAF-AH) and phosphatidase A2(PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extrac-ted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hy-drolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells ( HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase super-family, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688. 235 μmol/L and 0. 976/s, but its PLA2 activity was relatively weak. Expres-sion of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0. 05) and the secretion of LA_2144 gene product could be detec-ted. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activi-ty, which might involve in the hemorrhage and inflammatory response in leptospirosis.

Maturação seca e úmida da carne bovina e seus efeitos na qualidade sensorial e microbiológica

O perfil do consumidor brasileiro se tomou mais exigente em relação à qualidade da carne. Entre os atributos que fazem parte dessa definição, as características de palatabilidade são de suma importância para a avaliação de consumidor e podem ser melhoradas por meio da maturação. Essa técnica consiste em armazenar a carne em câmaras frias, sob condições controladas de temperatura, umidade relativa e velocidade do ar, por um período de tempo, a fim de que enzimas endógenas promovam mudanças em sua estrutura. Os métodos de maturação seca e úmida podem interferir diferentemente em muitos atributos. Em razão disso, esta revisão bibliográfica objetivou comparar esses dois processos, frente a alguns dos parâmetros de palatabilidade, como maciez, sabor e aroma da carne bovina, além da qualidade microbiológica dos produtos. Conclui-se que os processos de maturação, úmido e seco, melhoram a maciez da carne na mesma proporção, mas a maturação a seco possibilita o desenvolvimento de atributos de sabor mais desejáveis, quando comparado com a maturação úmida. Entretanto, os mecanismos responsáveis por esta melhoria ainda não são claros e precisam ser melhor investigados. De maneira geral a proliferação de micro-organismos é melhor controlada na maturação a seco, mas cuidados especiais devem ser tomados quando da etapa de toalete, remoção das superfícies ressecadas, para não haver contaminação cruzada.

The Brazilian consumer's profile has become more rigorous regarding to meat quality. Among the attributes that are part of definition of quality, palatability characteristics are the main concern for consumer evaluation and may be improved by aging. This technique consists in storing the meat in cold storage under controlled conditions of temperature, relative humidity and air flow during a period of time in order to promote changes in its structure by the activity of endogenous enzymes. The dry-aging and wet-aging differ in many attributes. Therefore, this review proposes to compare those two processes regarding to some of their palatability parameters such as tenderness, flavor and aroma in beef, in addition to verify the microbiological quality of the products. lt is concluded that the dry aging process is simple, but requires special care to meet desired sensory results and ensure microbiological safety.

Maturação seca e úmida da carne bovina e seus efeitos na qualidade sensorial e microbiológica

O perfil do consumidor brasileiro se tornou mais exigente em relação à qualidade da carne. Entre os atributos que fazem parte dessa definição, as características de palatabilidade são de suma importância para a avaliação do consumidor e podem ser melhoradas por meio da maturação. Essa técnica consiste em armazenar a carne em câmaras frias, sob condições controladas de temperatura, umidade relativa e velocidade do ar, por um período de tempo, a fim de que enzimas endógenas promovam mudanças em sua estrutura. Os métodos de maturação seca e úmida podem interferir diferentemente em muitos atributos. Em razão disso, esta revisão bibliográfica objetivou comparar esses dois processos, frente a alguns dos parâmetros de palatabilidade, como maciez, sabor e aroma da carne bovina, além da qualidade microbiológica dos produtos. Conclui-se que os processos de maturação, úmido e seco, melhoram a maciez da carne na mesma proporção, mas a maturação a seco possibilita o desenvolvimento de atributos de sabor mais desejáveis, quando comparado com a maturação úmida. Entretanto, os mecanismos responsáveis por esta melhoria ainda não são claros e precisam ser melhor investigados. De maneira geral a proliferação de micro-organismos é melhor controlada na maturação a seco, mas cuidados especiais devem ser tomados quando da etapa de toalete, remoção das superfícies ressecadas, para não haver contaminação cruzada

The Brazilian consumer's profile has become more rigorous regarding to meat quality. Among the attributes that are part of definition of quality, palatability characteristics are the main concern for consumer evaluation and may be improved by aging. This technique consists in storing the meat in cold storage under controlled conditions of temperature, relative humidity and air flow during a period of time in order to promote changes in its structure by the activity of endogenous enzymes. The dry-aging and wet-aging differ in many attributes. Therefore, this review proposes to compare those two processes regarding to some of their palatability parameters such as tenderness, flavor and aroma in beef, in addition to verify the microbiological quality of the products. It is concluded that the dry aging process is simple, but requires special care to meet desired sensory results and ensure microbiological safety

Isolamento e perfil enzimático de cães e gatos com dermatofitose atendidos em hospitais veterinários do Recife, Pernambuco / Isolation and enzymatic profile of dogs and cats with dermatofitosis attended at veterinary hospitals in Recife, Pernambuco, Brazil

Os dermatófitos são fungos que podem causar infecções superficiais da pele, cabelo e unhas em humanos e animais. As espécies de dermatófitos mais frequentemente isoladas dos cães e gatos afetados por micoses são Microsporum gypseum e principalmente Microsporum canis. O papel crucial durante o processo de infecção é a produção de enzimas extracelulares essenciais para a invasão e estabelecimento do agente patogênico no tecido do hospedeiro. O objetivo deste trabalho foi isolar dermatófitos de cães e gatos e avaliar o perfil enzimático dos isolados obtidos. Amostras de pelos e escamas epidérmicas foram coletadas de cães e gatos em instalações veterinárias em Recife/PE, e os isolados foram identificados com base nas características macroscópicas e microscópicas. A análise qualitativa das enzimas urease, protease, lipase, colagenase e fosfolipase foi avaliada a partir dos dermatófitos isolados. Durante 10 meses, um total de 106 animais, que compreendeu de 99 cães e sete gatos com sinais clínicos, independentemente do sexo e raça foram avaliados. Apenas oito animais foram confirmados com dermatofitose, principalmente cães (n=7), sendo seis afetados por M. canis e um por M. gypseum, a raça mais afetada foi Yorkshire (n=3). No entanto, apenas um gato foi confirmado com M. canis. Não foi observada predisposição relacionada ao sexo quanto à ocorrência de dermatofitose nos cães e gatos avaliados. Os dermatófitos isolados apresentaram perfis semelhantes para as enzimas urease, lipase, protease, fosfolipase e colagenase, característica importante em infecções patogênicas. O diagnóstico clínico destas zoonoses com base na confirmação microbiológica e uma compreensão dos mecanismos subjacentes é de grande importância para o tratamento e prevenção de doenças fúngicas em animais.(AU)

Dermatophytes are fungi that can cause superficial infections of the skin, hair and nails in man and animals. The most frequent dermatophyte species isolated from dogs and cats are Microsporum gypseum, most notably Microsporum canis. The crucial role during the infection process is the production of extracellular enzymes essential for the invasion and establishment of the pathogen in the host tissue. The objective of this research was to isolate dermatophytes from dogs and cats and evaluate the enzymatic profile of the isolates obtained. Hair samples and epidermal scales were collected from dogs and cats in veterinary facilities in Recife-PE, and the isolates were identified based on macroscopic and microscopic characteristics. The qualitative analysis of the enzymes urease, protease, lipase, collagenase and phospholipase was evaluated from the isolated dermatophytes. During 10 months, a total of 106 animals, comprising of 99 dogs and seven cats with clinical signs, regardless of sex and race were evaluated. Only eight animals were confirmed with dermatophytosis, mostly dogs (n=7), being six affected by M. canis and one by M. gypseum, the race most affected was Yorkshire (n=3). However, only one cat was confirmed with M. canis. No sex-related predisposition was observed regarding the occurrence of dermatophytosis in dogs and cats evaluated. Isolated dermatophytes showed similar profiles for the enzymes urease, lipase, protease, phospholipase and collagenase, important characteristic for pathogenic infections. The diagnosis of this zoonosis based on microbiological confirmation and a better understanding of the underlying mechanisms is of great importance for the treatment and prevention of fungal diseases in animals.(AU)

Filamentous fungi producing enzymes under fermentation in cassava liquid waste / Fungos filamentosos produtores de enzimas sob fermentação em manipueira

The conversion of agroindustrial residues by microorganisms has been explored from fermentative processes to obtain several bioactive molecules. The objective of this work was to isolate and select filamentous fungi present in cassava liquid waste for the production of amylase, carboxymethylcellulose (CMCase), pectinase and xylanase using the same residue as induction substrate in fermentative processes. A total of 65 filamentous fungi were isolated and qualitative tests indicated that approximately 86% of these strains were able to produce at least one of the enzymes and 32% capable of producing the four enzymes. Fermentation assays in cassava liquid residue-containing medium showed 6 fungal lines as potential enzyme producers. The maximum activities of pectinase, xylanase, amylase and CMCase were respectively observed at 96 hours of fermentation by the strain by the strain Aspergillus sp. B5C; at 120 hours (163.6 ± 0.13 nKat mL-1), by Aspergillus sp. B4I; at 144 hours (99.8 ± 0.24 nKat mL-1), by Penicillium sp. B3A; and at 48 hours (55.5 ± 0.21 nKat mL-1), by Aspergillus sp. B4O. These results suggest that cassava liquid waste was source of filamentous fungi producing amylase, CMCase, pectinase and xylanase, as well as a promising alternative substrate for bioprocesses aiming the production of enzymes.

A conversão de resíduos agroindustriais por micro-organismos tem sido explorada a partir de processos fermentativos para obtenção de diversas moléculas bioativas. O objetivo deste trabalho foi isolar e selecionar fungos filamentosos presentes em manipueira para produção de amilase, carboximetilcelulase (CMCase), pectinase e xilanase utilizando o próprio resíduo como substrato indutor. Um total de 65 fungos filamentosos foi isolado e testes qualitativos indicaram que, aproximadamente, 86% dessas linhagens foram hábeis em produzir pelo menos uma das enzimas e 32% capazes de produzir as quatro enzimas. Ensaios fermentativos em meio contendo manipueira apontaram 6 linhagens fúngicas como potenciais produtoras de enzimas. As atividades máximas de pectinase, xilanase, amilase e CMCase foram observadas, respectivamente, às 96 horas de fermentação (67.4 ± 0,6 nKat mL-1), pela linhagem Aspergillus sp. B5C; às 120 horas (163.6 ± 0,13 nKat mL-1), por Aspergillus sp. B4I; às 144 horas (99.8 ± 0,24 nKat mL-1), por Penicillium sp. B3A; e às 48 horas (55.5 ± 0,21 nKat mL-1), por Aspergillus sp. B4O. Estes resultados sugerem a manipueira como fonte de fungos filamentosos produtores de amilase, CMCase, pectinase e xilanase, além de um promissor substrato alternativo para bioprocessos visando a produção dessas enzimas.