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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.
Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Podophyllotoxin , Phylogeny , Cloning, MolecularABSTRACT
Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.
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Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.
Subject(s)
Bridged-Ring Compounds , Imidazoles , Ornithine , Ornithine Decarboxylase , Ornithine Decarboxylase Inhibitors , PutrescineABSTRACT
O artigo objetiva contribuir com o desenvolvimento teórico e metodológico no campo do trabalho em Educação. Discute a situação dos professores com contratos de prestação de serviço temporário, destacando a importância de os docentes analisarem coletivamente os diferentes modos como empreendem sua atividade de trabalho na perspectiva da produção de autonomia e criação. Os recursos metodológicos propostos apoiam-se em conceitos e princípios preconizados na Ergologia e na Clínica da Atividade. Considera-se que o método é resultado de uma construção conjunta com aqueles que demandam a transformação das escolas em seu próprio fazer cotidiano, produzindo inflexões no que tenta paralisar e limitar a inventividade no trabalho. Nesse sentido, trata-se de atuar na contramão dos processos de precarização do trabalho, indagando o que está instituído: O que temos feito de nós mesmos, para onde desejamos ir, quais são nossas apostas ético-políticas?
The paper aims at contributing to the theoretical and methodological development in the education work field. It discusses the situation of teachers provided with temporary service contracts by highlighting the importance of teachers collectively analyze the different ways in which they undertake their labor activity from the perspective of production autonomy and creation. The proposed methodological sources rely on concepts and principles advocated in Ergology and Clinical Activity. It is proposed that the method is the result of a joint construction with those who demand the transformation of schools in their own daily tasks, producing inflections in trying to paralyze and limit the ingenuity at work. In this sense, it is acting against the precariousness process of work by asking what is instituted: What have we done of ourselves, where do we want to go, and what are our ethical and political stakes?
El trabajo tiene la meta de contribuir con el desarrollo teórico y metodológico de lo campo de trabajo en la educación. Discute la situación de los profesores con provisión de contractos de servicios temporales, destacando la importancia de los maestros analizaren colectivamente las diferentes formas que se llevan a cabo su actividad laboral desde la perspectiva de la autonomía de producción y creación. Las características metodológicas se basan en conceptos y principios defendidos en la Ergología y la Clínica de la Actividad. Se propone que el método es el resultado de una construcción conjunta con los que exigen la transformación de las escuelas en sus tareas diarias, produciendo inflexiones en el intento de paralizar y limitar el ingenio en el trabajo. En este sentido, se está actuando en contra del proceso de precarización del trabajo haciendo lo que se instituyó: ¿Lo que hemos hecho nosotros mismos?, ¿dónde queremos ir?, ¿cuáles son nuestras apuestas éticas y políticas?
Subject(s)
Psychology , Faculty , Psychology, Educational , Work , Workload/psychology , EducationABSTRACT
Objective To isolate and culture the primary cerebellar granulosa cells(CGNs)of SD rats and evaluate the activity of botulinum neurotoxin A(BoNT/A)based on CGNs.Methods CGNs of 6-to 8-days-old SD rats were isolated and cultured.After 5-7 d,the cells were treated with BoNT/A.The activity of the toxin was evaluated with immunofluo-rescence,and the relationships between the activity and dose of the toxin were analyzed with Western blotting.Results and Conclusion CGNs Of SD rats were successfully cultured,a method for evaluating the activity of BoNT/A was established at the level of primary nerve cells,and the relationships between the activity and dose of toxin were analyzed.This study provides a tool for further detailing the biochemical mechanism of BoNT /A.
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Introdução: A proposta de intervenções por meio de gameterapia se aplica nos domínios da atuação do terapeuta ocupacional em diferentes contextos de prática. Objetivo: Este estudo teve por objetivo analisar jogos do videogame Nintendoï Wii, visando à sua utilização como atividade terapêutica para idosos. Método: Foram selecionados 15 minijogos do Wii Party, tendo como critérios o tempo de execução e o nível de complexidade da atividade para a exequibilidade do uso com idosos. A análise foi realizada com base no referencial teórico adotado pela Associação Americana de Terapia Ocupacional, composta por sete passos: I) identificação da atividade; II) tempo e sequenciamento da atividade; III) demandas sociais, objetos e espaço; IV) funções corporais requeridas; V) estruturas corporais requeridas; VI) ações requeridas; VII) desempenho de habilidade e análise para intervenção. Resultados: Foram identificadas possibilidades de indicações relacionadas às funções cognitivas e habilidades específicas, evidenciando-se, dessa forma, o potencial do jogo para uso com idosos que apresentem queixas relacionadas a funções cognitivas. Considerou-se também a importância de monitoramento do comportamento do usuário, propondo-se uma ficha para acompanhamento e avaliação do seu desempenho na atividade. Conclusão: Este estudo ofereceu indicadores para a sistematização do monitoramento de uso de jogos do videogame Nintendoï Wii como atividade de intervenção da terapia ocupacional, contribuindo para a prática clínica na atenção à população idosa e seus desdobramentos na formação do terapeuta ocupacional, e para a pesquisa na área da saúde do idoso.
Introduction: Intervention through gametherapy is applied to Occupational Therapy domains in different practical contexts. Objective: This study aimed to analyze the videogame Nintendoï Wii, with a view to its use as a therapeutic activity for elderly population. Method: 15 mini-games in Wii Party were selected based on criteria of execution time and the activity level of complexity, for the feasibility of use with the elderly. The analysis was based on the theoretical framework adopted by the American Association of Occupational Therapy, and consists of seven steps: I) activity identification; II) time and sequencing; III) social demands, objects and space; IV) body functions required; V) body structures required; VI) required actions, VII) skill performance; analysis for intervention. Results: indications of possibilities related to cognitive functions and specific skills were identified, evidencing thus, the game potential for use with older people who have cognitive functions problems. It is also considered the importance of monitoring user behavior, proposing a form for monitoring and evaluation of their performance in the activity. Conclusion: This study provides indicators for the systematic monitoring of Nintendoï Wii videogame games use as an intervention activity of occupational therapy, contributing to clinical practice in the care of the elderly population and its consequences in the formation of occupational therapist and research in the elderly health.
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Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg.
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Introducción: la frecuencia del pulso es un indicador directo del estado del sistema cardiovascular, además de ser un indicador indirecto de la energía gastada en la ejecución de una tarea. El pulso de una persona es el número de pulsaciones registradas en una arteria periférica por unidad de tiempo, que se manifiesta como una onda de presión que se mueve a lo largo de los vasos sanguíneos, los cuales son flexibles. "En las grandes ramas arteriales, su velocidad es de 7 a 10 m/s y en las arterias pequeñas, de 15 a 35 m/s". Materiales y métodos: el fin de este estudio fue evaluar la frecuencia cardíaca, utilizando la técnica de registro de la frecuencia del pulso, el consumo de oxígeno y la observación de la actividad de trabajo para la estimación de la carga de trabajo en una tarea de manipulación de carga para tres situaciones: levantar/trasladar/depositar; antes, durante y después de la tarea, se registra la frecuencia del pulso para 24 jóvenes voluntarios (10 mujeres y 14 hombres) en condiciones de laboratorio. Simultáneamente, se realizó un registro del gesto de trabajo y de las estrategias de levantamiento, movilización y depósito de la carga. Resultados: se observó un incremento entre la FP inicial y final en los dos grupos y para las dos tareas; se registra, igualmente, una diferencia en el aumento de las pulsaciones para la carga de 17,5. El 75% de los participantes experimenta un incremento de la FP por encima de 100 lat./min. Para los 25 kg, los valores registrados indican valores superiores a 114 lat./min y, para los 17,5 kg, valores superiores a 128 lat./min. Conclusión: la frecuencia del pulso es un método que se recomienda por su simplicidad de uso para el personal operativo, supervisores y gerentes, así como para los ingenieros industriales no entrenados en el método fisiológico, también puede ser utilizado por higienistas industriales.
Introduction: The pulse rate is a direct indicator of the state of the cardiovascular system, in addition to being an indirect indicator of the energy expended in performing a task. The pulse of a person is the number of pulses recorded in a peripheral artery per unit time; the pulse appears as a pressure wave moving along the blood vessels, which are flexible, "in large arterial branches, speed of 7-10 m/s in the small arteries, 15 to 35 m/s". Materials and methods: The aim of this study was to assess heart rate, using the technique of recording the frequency of the pulse, oxygen consumption and observation of work activity in the estimation of the workload in a load handling task for three situations: lift/transfer/deposit; before, during and after the task the pulse rate is recorded for 24 young volunteers (10 women and 14 men) under laboratory conditions. We performed a gesture analysis of work activity and lifting and handling strategies. Results: We observed an increase between initial and final FP in both groups and for the two tasks, a difference is also recorded in the increase in heart rate of 17.5 for charging 75% of the participants experienced an increase in FP above 100 lat./min. Par 25 kg, registered values indicate greater than 114 lat./min and 17.5 kg than 128 lat./min values. Conclusion: The pulse rate method is recommended for its simplicity of use for operational staff, supervisors and managers and industrial engineers not trained in the physiology method can also be used by industrial hygienists.
Introdução: a frequência do pulso é um indicador direto do estado do sistema cardiovascular além de ser um indicador indireto da energia gastada na execução de uma tarefa. O pulso de uma pessoa é o número de pulsações registradas em uma artéria periférica por unidade de tempo; o pulso se manifesta como uma onda de pressão que se move ao longo dos vasos sanguíneos, que são flexíveis. "Nos grandes ramos arteriais, sua velocidade é de 7 a 10 m/s e nas artérias pequenas de 15 a 35 m/s". Materiais e métodos: o fim deste estudo foi avaliar a frequência cardíaca, utilizando a técnica de registro da frequência do pulso, o consumo de oxigeno e a observação da atividade de trabalho para a estimação da carga de trabalho em uma tarefa de manipulação de carga para três situações: levantar/trasladar/depositar; antes, durante e depois da tarefa se registra a frequência do pulso para 24 jovens voluntários (10 mulheres e 14 homens) em condições de laboratório. Simultaneamente se realizou um registro do gesto de trabalho e das estratégias de levantamento, mobilização e depósito da carga. Resultados: observou-se um incremento entre a FP inicial e final nos dois grupos e para as duas tarefas; registra-se igualmente, uma diferença no incremento das pulsações para a carga de 17,5 o 75% dos participantes experimenta um incremento da FP por cima de 100 lt/min. Para os 25 KG, os valores registrados indicam valores superiores a 114 lt/ min e para os 17,5 KL g valores superiores a 128 l/min. Conclusão: a frequência do pulso é um método que se recomenda por sua simplicidade de uso para o pessoal operativo, supervisores e gerentes, assim como os engenheiros industriais não treinados no método isiológico; também pode ser utilizado por higienistas industriais.
Subject(s)
Humans , Heart Rate , Oxygen Consumption , Data Collection , Workload , Observation , Energy MetabolismABSTRACT
Objective:To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry ( UPLC-MS/MS) method to determine CYP2C9 activity in vitro. Methods:An ACQUITY UPLC? BEH C18 (100 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water ( containing 0. 1% formic acid and 0. 5%ammonia water) (40∶60, v/v). The flow rate was 0. 2 ml·min-1. Chlorpropamide was used as the internal standard. The MS condi-tions were as follows:ESI with positive ion detection mode. Self-prepared CYP2C9?1, ?2, ?3 and ?13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000g, the organic layer was then dried using nitrogen, the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS/MS. Results: The reten-tion time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl-1(r=0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0. 01 ng·μl-1 with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9?2,?3 and?13 after tol-butamide was incubated with CYP2C9?1,?2,?3 and?13 was 47. 3%, 11% and 0. 3% of wild type CYP2C9?1. Conclusion:The method is simple and stable, and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.
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PURPOSE: The purpose of this study was to estimate the cost of dental implant using the bottom-up approach with the current data from dental clinics. MATERIALS AND METHODS: In this study, direct and indirect costs required for each treatment were calculated using the bottom-up approach. In the bottom-up costing, the average monthly total cost of dental clinic includes labor and material costs, administrative expenses, medical malpractice costs, and opportunity costs of invested capital. For the dental implant cost components, those include direct costs (labor costs, laboratory costs, material costs, depreciation or other operating costs), indirect costs (administrative costs), and the opportunity costs of investment for dental clinic. RESULTS: Dental implant costs of metal crown, porcelain crown and over-denture were 1,449,000 won, 1,583,000 won, and 2,471,000 won respectively. The proportion of cost components was as follows. The labor cost were 50%, and material, administrative and other cost were 33%, 15% and 2%, respectively. For direct, indirect and investment cost, the ratio were 83%, 15% and 2%, respectively. CONCLUSION: The labor costs were evaluated to comprise largest proportion (about 50%, 730,000 won). Dental implant cost using Bottom-up costing was 1,450,000 won for metal crown and 1,580,000 won for porcelain crown.
Subject(s)
Costs and Cost Analysis , Crowns , Dental Clinics , Dental Implants , Dental Porcelain , Depreciation , Investments , MalpracticeABSTRACT
Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue.Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results The expression plasmid pET32a-Rv0091 was constructed and expressed in E.coli.BL21trxB,and the optimum expression system was conformed.The purity of the recombinant protein was more than 95％.Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins.Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same.Enzyme assay showed the recombinant protein able to catalyze the substrate MTA.Enzymatic properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.
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Objective To clone the human mucin (MUC)5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. Methods The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software.After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced.By promoter deletion analysis, 3 promoter segments with diferent lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors.Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-кB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Results Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end.The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully.Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P＜0.05 vs. contro1) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. Conclusion This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression.There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.