Analysis of clinical significance and prognostic impact of TET2 single nucleotide polymorphism I1762V in patients with acute myeloid leukemia / 中华血液学杂志

Objective: This study aimed to investigate the clinical and prognostic significance of TET2 single nucleotide polymorphism I1762V in patients with acute myeloid leukemia (AML) . Methods: The high-throughput sequencing method was used to sequence 58 hematological tumor-related genes in bone marrow samples from 413 patients with AML. TET2 I1762V and other somatic mutations were annotated and compared with patients' clinical information and prognosis. Results: I1762V was found in 154 patients with AML, which was significantly different from the general population in NyuWa Chinese Population Variant Database (χ(2)=72.4, P<0.001) . I1762V was not related to sex, age, and karyotype of patients with AML (P>0.05) . Patients with I1762V had a significantly higher proportion of NPM1 and KIT gene mutations than others (P<0.001) . NPM1 and KIT mutations were mutually exclusive. The survival analysis results revealed that the overall survival (OS) and progression-free survival (PFS) of patients with AML with I1762V were significantly greater than those of wild-type patients (HR=0.57, P=0.030; HR=0.55, P=0.020) , whereas the OS and PFS in patients with AML with DNMT3A mutation (with or without I1762V mutation) were lower than those of wild-type patients (HR=1.79, P=0.030; HR=1.74, P=0.040) . Conclusion: TET2 SNP I1762V has been linked to AML. I1762V is a prognostic factor of patients with AML, which can be used to guide the treatment and evaluate the prognosis of AML.

Advances in bacterial Rieske non-heme iron ring-hydroxylating dioxygenases that initiate polycyclic aromatic hydrocarbons degradation / 生物工程学报

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants, which have received widespread attentions due to their carcinogenic and mutagenic toxicity. The microbial degradation of PAHs are usually started from the hydroxylation, followed by dehydrogenation, ring cleavage and step-by-step removal of branched chains, and finally mineralized by the tricarboxylic acid cycle. Rieske type non-heme iron aromatic ring-hydroxylating dioxygenases (RHOs) or cytochrome P450 oxidases are responsible for the conversion of hydrophobic PAHs into hydrophilic derivatives by the ring hydroxylation. The ring hydroxylation is the first step of PAHs degradation and also one of the rate-limiting steps. Here, we review the distribution, substrate specificity, and substrate recognition mechanisms of RHOs, along with some techniques and methods used for the research of RHOs and PAHs.

L-DOPA extradiol dioxigenase de amanita muscaria: revisão da sequência codificadora, expressão heteróloga, caracterização funcional e contextualização filogenética / Amanita muscaria L-DOPA extradiol dioxygenase: coding sequence review, heterologous expression, functional characterization, and phylogenetic context

Extradiol dioxigenases são enzimas que catalisam a clivagem oxidativa de ligações C-C entre grupos hidroxila fenólicos adjacentes utilizando catecóis como substratos. Esta classe de enzimas é bem caracterizada em bactérias, onde catalisam a degradação de compostos aromáticos. Na maioria das plantas Caryophyllales, como a beterraba, primavera e a maravilha, L-3,4-diidroxifenilalanina (L-DOPA) extradiol dioxigenases (DODAs) catalisam a clivagem oxidativa de L-DOPA na posição 4,5 gerando o ácido betalâmico, aldeído precursor das betalaínas, uma classe de pigmentos naturais que substitui as antocianinas na pigmentação dessas espécies. Alguns fungos basidiomicetos também produzem betalaínas, como o agário-das-moscas (Amanita muscaria). Nesse organismo, DODA é capaz de catalisar uma clivagem adicional na posição 2,3 da L-DOPA, formando muscaflavina, um isômero do ácido betalâmico que dá origem a uma outra classe de pigmentos naturais: as higroaurinas. Desde a caracterização do gene dodA, o qual codifica para a DODA de A. muscaria (AMAMU), não existem relatos na literatura que explorem a promiscuidade catalítica desta enzima, sua relação com outras linhagens de DODAs e a síntese quimioenzimática de betalaínas a partir desta enzima. Dessa forma, buscamos contextualizar as relações filogenéticas e funcionais entre AMAMU e diferentes linhagens de DODAs, bem como estabelecer um método que viabilize a clonagem, expressão heteróloga e caracterização funcional destaenzima. As análises filogenéticas revelaram que AMAMU possui uma evolução convergente com DODAs de plantas e bactérias e que, apesar de AMAMU ser funcionalmente homóloga à DODA da bactéria Escherichia coli, esta última apresenta homologia com DODAs de plantas. Logo, não há uma relação direta entre a sequência primária de DODAs e sua função. Nós também demonstramos que não há uma relação entre a expressão de transcritos de BvDODA1, e de seu parálogo BvDODA2, e a diferença de pigmentação entre variedades de beterrabas amarelas e vermelhas. A clonagem da sequência codificadora (CDS) publicada para o gene dodA de A. muscaria resultou na retenção do primeiro íntron, o que impedia a sua expressão. Então, uma nova CDS de 558 nucleotídeos foi proposta para este gene, a qual inclui um códon de início da tradução que se mantém na fase de leitura e codifica para uma proteína de 185 resíduos, 43 a menos que AMAMU. A expressão desta CDS resultou na proteína recombinante AmDODA, capaz de catalisar a síntese de ácido betalâmico e muscaflavina a partir de L-DOPA e D-DOPA. AmDODA possui um tamanho aproximado de 22 kDa, com um pH ótimo de atividade de 8,5 e uma constante de Michaelis (KM) de 3,7 ± 0,9 mmol L-1 e de velocidade máxima (Vmax) de 3,3 ± 0,4 µ mol min-1 mg-1. Sua utilização foi demonstrada na síntese quimioenzimática de betalaínas-modelo com potencial aplicação como sondas para microscopia confocal de fluorescência de dois fótons. Neste contexto, esta Tese explora os aspectos moleculares, bioquímicos e biológicos da DODA do fungo A. muscaria e traz importantes contribuições acerca da pigmentação por betalaínas na natureza

Extradiol dioxigenases are enzymes that catalyze the oxidative cleavage of C-C bonds between adjacent phenolic hydroxyl groups using catechols as substrates. This class of enzymes is well characterized in bacteria, where they catalyze the degradation of aromatic compounds. In most plants of the Order Caryophyllales, such as beet, paperflower and four o'clock flower, L-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases (DODAs) catalyze the oxidative 4,5-cleavage of L-DOPA generating the betalamic acid, an aldehyde precursor of the betalains, a class of natural pigments that replaces anthocyanins in the pigmentation of these species. Some basidiomycete fungi also produce betalains, such as the fly agaric (Amanita muscaria). In this organism, DODA is able to catalyze an additional 2,3-cleavage of L-DOPA, yielding muscaflavine, an isomer of betalamic acid that gives rise to another class of natural pigments: the hygroaurins. Since the characterization of the dodA gene, which encodes the A. muscaria DODA (AMAMU), there are no reports in the literature that explore the catalytic promiscuity of this enzyme, its relation to other DODAs and the chemoenzymatic synthesis of betalains from this enzyme. Thus, we seek to contextualize the phylogenetic and functional relationships between AMAMU and different DODA lineages, as well as to establish a method that enable the cloning, heterologous expression and functional characterization of this enzyme. Phylogenetic analysis revealed that AMAMU has a convergent evolutionwith plant and bacterial DODAs and that although AMAMU is functionally homologous to the DODA of the Escherichia coli bacteria, this latter is homologous to the plant DODAs. Therefore, there is no direct relationship between the primary sequence of DODAs and their function. We have also shown that there is no relationship between the expression of BvDODA1 transcripts, and its BvDODA2 paralogue, and the pigment difference between yellow and red beet varieties. Cloning of the published coding sequence (CDS) for the dodA gene of A. muscaria resulted in the retention of the first intron, which prevented its expression. Then, a new CDS of 558 nucleotides was proposed for this gene, which includes a translation start codon that remains in the open reading frame and encodes for a protein 185 residues long, 43 less than AMAMU. Expression of this CDS resulted in the recombinant AmDODA protein, able to catalyze the synthesis of betalamic acid and muscaflavine from L-DOPA and D-DOPA. AmDODA has an approximate size of 22 kDa, with an optimum activity pH of 8.5 and a Michaelis constant (KM) of 3.7 ± 0.9 mmol L-1 and a maximum velocity (Vmax) of 3.3 ± 0.4 µmol min-1 mg-1. Its use was demonstrated in the chemoenzymatic synthesis of betalains-model with potential application as probes for confocal microscopy of two-photon fluorescence. In this context, this thesis explores the molecular, biochemical and biological aspects of the DODA of the fungus A. muscaria and brings important contributions about the pigmentation by betalains in nature

Function analysis of anthocyanidin synthase from Morus alba L. by expression in bacteria and tobacco

Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

ABSTRACT Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

TET family dioxygenases and DNA demethylation in stem cells and cancers

The methylation of cytosine and subsequent oxidation constitutes a fundamental epigenetic modification in mammalian genomes, and its abnormalities are intimately coupled to various pathogenic processes including cancer development. Enzymes of the Ten-eleven translocation (TET) family catalyze the stepwise oxidation of 5-methylcytosine in DNA to 5-hydroxymethylcytosine and further oxidation products. These oxidized 5-methylcytosine derivatives represent intermediates in the reversal of cytosine methylation, and also serve as stable epigenetic modifications that exert distinctive regulatory roles. It is becoming increasingly obvious that TET proteins and their catalytic products are key regulators of embryonic development, stem cell functions and lineage specification. Over the past several years, the function of TET proteins as a barrier between normal and malignant states has been extensively investigated. Dysregulation of TET protein expression or function is commonly observed in a wide range of cancers. Notably, TET loss-of-function is causally related to the onset and progression of hematologic malignancy in vivo. In this review, we focus on recent advances in the mechanistic understanding of DNA methylation-demethylation dynamics, and their potential regulatory functions in cellular differentiation and oncogenic transformation.

Características de las residencias de terapia intensiva pediátrica de la República Argentina: Encuesta nacional / Characteristics of pediatric intensive care residency programs in Argentina: A national survey

Introducción. La residencia de terapia intensiva pediátrica (TIP) tiene pocos años de desarrollo en nuestro país. Conocer su situación brinda la posibilidad de establecer estrategias para contribuir al desarrollo y capacitación de profesionales. Objetivos. 1) Describir las características de las residencias de TIP del país. 2) Evaluar si existen características que se relacionen con una mayor ocupación de las vacantes. 3) Explorar la inserción laboral en el hospital formador de los residentes. Diseño. Descriptivo, observacional. Encuesta nacional. Criterios de inclusión. Residencias de TIP funcionales entre el 1/4/2014 y el 31/5/2014. Resultados. Se analizaron 31 residencias. Solo 11/31 tenían volumen de internación anual >400 pacientes. No había normas y/o criterios de atención en 9/31. En 17/31, el programa estuvo adecuado al marco de referencia nacional. Hubo 13/31 que no contaban con jefe ni instructor de residentes. Fueron acreditadas por el Ministerio de Salud 5/31. Hubo 65 vacantes; el número aumentó en los últimos 4 años; la ocupación disminuyó de 59% en 2009 a 30% en 2013. El 60% de los residentes tuvo inserción laboral en la TIP formadora. El análisis de regresión logística multivariado identificó la variable ingresos anuales > 400 pacientes como predictora independiente de ocupación de vacantes > 60%. Conclusiones. 1) Hay un déficit en la ocupación de cargos. 2) El número de residencias acreditadas es escaso. 3) Las unidades de cuidados intensivos pediátricos con mayor número de ingresos se asociaron a una mayor cobertura de vacantes. 4) Más de la mitad de los residentes se insertaron laboralmente en la TIP formadora.

Introduction. Pediatric intensive care residency programs have been in place in Argentina for just a few years. Knowing their status offers the possibility to establish strategies to help with professional development and training. Objectives. 1) To describe the characteristics of pediatric intensive care residency programs across Argentina. 2) To assess whether certain characteristics are related to a higher vacancy filling rate. 3) To assess job placement in the hospital where residents are trained. Design. Descriptive, observational study. National survey. Inclusion criteria. Pediatric intensive care residency programs in place between April 1st, 2014 and May 31st, 2014. Results. Thirty-one residency programs were analyzed. Only 11/31 had an annual hospitalization volume >400patients. There were no guidelines and/or criteria for care in 9/31. The program suited the national reference frameworkin17/31. There was no head ofresidents or resident trainer in 13/31. Only 5/31 had been certified by the Ministry of Health. There were 65 vacancies; this number increased in the past four years; vacancy filling rate decreased from 59% in 2009 to 30% in 2013. Sixty percent of residents got a job in the pediatric intensive care unit where they were trained. A multivariate logistic regression analysis identified the outcome measure annual hospitalization volume >400 patients as an independent predictor of vacancy filling rate >60%. Conclusions. 1) Vacancy filling is deficient. 2) The number of certified residency programs is scarce. 3) Pediatric intensive care units with a higher number of hospitalizations were associated with a higher vacancy filling rate. 4) More than half of residents got a job in the pediatric intensive care unit where they were trained.

Effect of cadmium on TET enzymes and DNA methylation changes in human embryonic kidney cell / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the expression changes of the demethylase TETs (Ten-eleven translocation enzymes) in human embryonic kidney cell (HEK293) exposed to high dose cadmium chloride (CdCl2), and to investigate the regulation effects of TETs on global genomic methylation.</p><p><b>METHODS</b>HEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 (cell counting kit-8) method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay.</p><p><b>RESULTS</b>CdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration (IC50) was 1.78 µmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group (2.0 µmol/L) showed vacuolar changes and fuzzy appearance. The level of TET1 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.13, 0.48 ± 0.12, 0.59 ± 0.16 and 0.95 ± 0.39, respectively (F = 182.89, P = 0.002); The level of TET2 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.12, 0.32 ± 0.02,0.31 ± 0.10 and 0.34 ± 0.07, respectively (F = 27.94, P < 0.001); The level of TET3 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.26 ± 0.10, 0.27 ± 0.11, 0.25 ± 0.11 and 0.28 ± 0.09, respectively (F = 1.76, P = 0.036). The interaction effect existed between exposure time and doses of TET1 mRNA, TET2 mRNA and TET3 mRNA (F values were 32.94, 23.04 and 13.78, respectively; P values were < 0.001, 0.041 and < 0.001, respectively). Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels. In 24 h (55.01 ± 3.62)%, 48 h (48.31 ± 8.99)%, 72 h (48.76 ± 6.60)%, the DNA methylation had significant differences (F = 18.50, P < 0.001); In groups of 0.0 µmol/L (55.29 ± 2.83)%, 0.5 µmol/L (55.35 ± 3.11)%, 1.0 µmol/L (48.58 ± 6.40)% and 2.0 µmol/L (43.56 ± 7.89)%, the differences of DNA methylation had significant differences (F = 7.03, P = 0.048); the effect of interaction was also existed (F = 2.73, P = 0.043).</p><p><b>CONCLUSION</b>In the short term exposure to CdCl2, the levels of TETs mRNA and protein showed a trend of increase according to the exposure time and dose, and the methylation level of whole genomic DNA was also altered. The demethylase TETs may play a role in regulating the genomic methylation level of HEK293 exposed to cadmium.</p>

Instituto de Radium de Minas Gerais: vanguarda da radioterapia no Brasil, 1923-1935 / Minas Gerais Radium Institute: at the forefront of radiotherapy in Brazil, 1923-1935

Este artigo propõe estudar os primeiros 12 anos de existência do Instituto de Radium de Minas Gerais, fundado em 1922. Sua atuação na luta contra o câncer no Brasil, ainda pouco conhecida, começa a ser esboçada pelo estudo de documentação institucional inédita. Através de um banco de dados elaborado com informações constantes em seu livro de registro de pacientes, foram feitos levantamentos estatísticos dos tipos de câncer e das formas de tratamento existentes entre 1923 e 1935. Esse livro faz parte de um conjunto de outros cinco recentemente descobertos no Centro de Memória da Medicina/UFMG. A documentação permite resgatar os primórdios das intervenções de radioterapia no país e acompanhar seu desenvolvimento e a influência exercida por esse hospital modelo.

This article proposes to study the first 12 years of the Minas Gerais Radium Institute, founded in 1922. Its work in the fight against cancer in Brazil, albeit still little known, is coming to light as its institutional documents are studied. A database has been prepared using information from its patient register, based on which statistical analyses have been done to identify the types of cancer and treatments available there between 1923 and 1935. This register is one of five recently unearthed at the Medicine Memory Center of the Universidade Federal de Minas Gerais. Through them, the earliest experiments in radiotherapy in Brazil can be reconstituted, and its development and the influence of this model hospital can be mapped out.

Properties of catechol 1, 2 dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O) by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L-1 of anthracene (PAH). The optimum conditions of pH (4.0-9.0), temperature (5-70 ºC), reaction time (10-90 min) and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 ºC, 20 ºC higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract

Molecular modeling of 2-nitropropane dioxygenase domain of Mycobacterium tuberculosis H37Rv and docking of herbal ligands.

The 3D structure of enoyl reductase (ER) domain generated by the SWISS MODEL server contains the 2-nitropropane dioxygenase (2NPD) structure displaying the TIM barrel fold. Though TIM barrel fold is made up of both main and inserted domains, in our study, we could only predict the structure of the main domain, which had central barrel of eight β-strands surrounded by eight α-helices. Superimposition of the 2NPD region of ER domain of Mycobacterium tuberculosis H37Rv on to the corresponding region of 2UVA_G revealed a good structural alignment between the two, suggesting this template to be a good structural homologue. Among various herbal ligands that were screened as inhibitors, daucosterol was found to bind in closest proximity to the flavin mono nucleotide (FMN) binding site with the lowest docking energy

Cloning, molecular characterization and expression of acireductone dioxygenase (ARD) gene from Salvia miltiorrhiza / 中国中药杂志

<p><b>OBJECTIVE</b>To study the acireductone dioxygenase (designated as SmARD) gene of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression and response expression on stress in shoot.</p><p><b>METHOD</b>SmARD gene was obtained by sequencing cDNA library constructed by us. BLAST was used for alignment, ORF finder software was applied to find open reading frame, prosite was used to analyze the protein characterization. Semi-quantitative RT-PCR was used to detect the gene expression level.</p><p><b>RESULT</b>The full -length cDNA of SmRAD was 688 bp long with a 591 bp ORF (open reading frame) that putatively encoded a polypeptide of 196 amino acids; with a predicted molecular mass of 23.27 kDa. The deduced amino acid sequence of SmRAD of gene shared high homology with other known RADs. Semi-quantitative RT-PCR analysis indicated that SmRAD was constitutively expressed in roots, stems, flower and leaves of S. miltiorrhiza, with the high expression in roots. In addition, SmRAD expression level under different stress condition was also analyzed in root, including signaling components for plant defence responses, such as methyl jasmonate, salicylic acid and ABA, as well as drought, cold and salt abiotic stress. The expression of SmRAD was suppressed by water deficit treatment for 3 d, 150 mmol x L(-1) NaCl, 4 degrees C cold and 100 mmol x L(-1) ABA treatment for 1 d, but induced by 100 mmol x L(-1) MJ and 10 mmol x L(-1) ETH.</p><p><b>CONCLUSION</b>A novel SmARD gene was cloned from S. miltiorrhiza. This study will enable us to further understand the role of SmARD in the defense response under different abiotic stress and in synthesis of active cmpounds in S. miltiorrhiza at molecular level.</p>

Papel imunossupressor da indoleamina 2, 3 - dioxigenase na hanseníse / Immunosuppressive role of indoleamine 2, 3 - dioxygenase in leprosy

Os mecanismos que levam a ausência de resposta imune celular no pólo lepromatoso da hanseníase frente ao Mycobacterium leprae permanecem obscuros. Estudos anteriores mostraram que fatores secretados pelos macrófagos de pacientes lepromatosos inibem a proliferação de linfócitos de indivíduos saudáveis, sugerindo que fatores endógenos produzidos por células da linhagem mielóide podem exercer uma atividade supressora nas células T. Recentemente, diversos estudos têm demonstrado a participação da enzima indoleamina 2,3 dioxigenase (IDO) na supressão das células T, indicando que mudanças bioquímicas devido ao catabolismo do triptofano têm efeitos na proliferação dessas células. No presente trabalho nós demonstramos que os pacientes com a forma clínica lepromatosa apresentam uma maior expressão de IDO em biópsias de pele e maior atividade de IDO no soro quando comparados com pacientes com a forma tuberculóide e que o M. leprae induz a expressão e a atividade de IDO em monócitos de indivíduos saudáveis e de pacientes lepromatosos. Além disso, observamos que o M. leprae induz aumento na atividade de IDO nas células dendríticas derivadas de monócitos de indivíduos saudáveis e que esta atividade aumentada de IDO contribui para o aumento percentual de linfócitos expressando a molécula supressora CTLA-4, sugerindo que IDO participe na anergia T antígeno específico observada em pacientes com a forma lepromatosa da doença.

Regulação cruzada entre peroxidases e indolamina 2.3 dioxigenase no controle da metabolização do triptofano / Peroxidases and indoleamine 2.3 dioxygenase crosstalk modulating tryptophan metabolization

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...

Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...

Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation / Isolamento e caracterização de uma nova cepa de Stenotrophomonas maltophilia com várias dioxigenases para degradação de hidrocarbonetos monocíclicos

A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2- dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases' types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination.

Uma bactéria Gram-negativa, denominada KB2, foi isolada de lodo ativado, verificando-se ser capaz de utilizar substratos aromáticos com única fonte de carbono e energia. Com base nas características morfológicas e físico-químicas, e na análise da sequencia do gene 16SrRNA, esta bactéria foi identificada como Stenotrophomonas maltophilia. Entre as diversas cepas de S. maltophilia já descritas, essa cepa é a primeira com atividade de três tipos de dioxigenases, dependendo da estrutura do indutor. As células cultivadas em benzoato e catecol apresentaram atividade de catecol 1,2-dioxigenase principalmente. A atividade de 2,3-dioxigenase foi detectada após indução com fenol. Após incubação com ácidos 4-hidrobenzoico, ácido protocatecuico evanílico, encontrou-se protocatecuato 3,4-dioxigenase no extrato celular. Devido ao amplo espectro de atividade das diferentes dioxigenases de S. maltophilia KB2, esta cepa parece ser uma ferramenta poderosa e útil para o biotratamento de efluentes e descontaminação do solo.

Enzymatic characteristics of quercetinases from some indigenous Aspergillus flavus strains.

Five indigenous Aspergillus flavus strains MTCC-2206, 1884, 1883, 1783 and 2456 were screened for the secretion of quercetinase. Fungal strains MTCC-2206, 1884, 1883, and 1783 were found to secrete the quercetinase in the range of 0.24-0.36 enzyme unit/mL of the culture medium, while MTCC-2456 secreted only 0.04 enzyme unit/mL. The enzymatic characteristics of quercetinase were determined. The Km values using quercetin as the substrate were 12.5 microM, 14.0 microM, 12.5 microM and 13.0 microM for the quercetinase produced by MTCC-2206, 1884, 1883 and 1783, respectively. The pH optima for the above enzymes were 6.5, 6.5, 6.0 and 6.0 and temperature optima were 45, 40, 45 and 50 degrees C, respectively. The partial purification from only one strain MTCC-2206 was achieved (nearly 3-fold purification).

Microbial biosynthesis and biotransformation of indigo and indigo-like pigments / 生物工程学报

Indigo and indigo-like pigments are widely used in the industry of textile, food and medicine. Now people pays more and more attention to developing an alternative method of indigo production which is "environment-friendy", especially microbial biosynthesis of indigo. Many microorganisms involved in the biosynthesis of indigo have been isolated and characterized, and monooxygenase and dioxygenase have been identified to catalyze indigo biosynthesis. Some genes encoding for these enzymes have been cloned and used to construct "engineering bacteria". With this kind of bacteria, more efficient fermentation systems for indigo production have been exploited. In the meantime, biotransformation of the indigo produced by microorganisms has been under investigation. These progresses will bring us a greener method of indigo and indigo-like pigments production.

Cloning and functional study of a novel aromatic-ring-hydroxylating dioxygenase gene / 南方医科大学学报

The aromatic-ring-hydroxylating dioxygenase is a key enzyme that initiates the biodegradation of polycyclic aromatic hydrocarbons in bacteria. In the present study, a novel dioxygenase sequence was cloned from Terrabacter sp. FLO using a genome walking method. The dioxygenase was cloned into pET17 and actively expressed in E.coli BL21 (DE3) in co-expression with electron transfer chain proteins. The recombinant dioxygenase was found to transform phenanthrene, fluorene, pyrene and fluoranthene into the cis-dihydrodiol metabolites.

Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

Abscisic acid (ABA) regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (https://forests.esalq.usp.br). A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED), the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g), which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays) NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana) NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process