Identification of paeoniflorin from Paeonia lactiflora pall. As an inhibitor of tryptophan 2,3-dioxygenase and assessment of its pharmacological effects on depressive mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The radix of Paeonia lactiflora Pall. (PaeR) is a traditional Chinese medicine (TCM) clinically used for treating depression. Although it has been established that PaeR can protect the liver and alleviate depressive-like behaviors, its bioactive chemicals and antidepressant mechanism remain unclear. Our pilot study showed that PaeR reduced the expression of the L-tryptophan- catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) in the livers of stress-induced depression-like mice. AIM OF THE STUDY: This study aimed to screen potential TDO inhibitors from PaeR and investigate the potential therapeutic use of TDO inhibition for treating depression. MATERIALS AND METHODS: Molecular docking, magnetic ligand fishing, and secrete-pair dual luminescence assay were conducted for in vitro ligand discovery and high-throughput screening of TDO inhibitors. Stable TDO overexpression was achieved in HepG2 cell lines to evaluate the TDO inhibitory activities of drugs in vitro by RT-PCR and Western blot analyses of TDO at mRNA and protein levels. In vivo validation of TDO inhibitory potency and evaluation of TDO inhibition as a potential therapeutic strategy for major depressive disorder (MDD) were performed using mice subjected to "3 + 1″ combined stresses for at least 30 days to induce depression-like behaviors. A well-known TDO inhibitor, LM10, was evaluated in parallel. RESULTS: The PaeR extract significantly ameliorated depressive-like behaviors of stressed mice, attributed to inhibition of TDO expression and tryptophan modulation metabolism. After a comprehensive analysis of molecular docking, ligand fishing, and luciferase assay, paeoniflorin was screened as a TDO inhibitor from the PaeR extract. This compound, structurally different from LM10, potently inhibited human and mouse TDO in cell- and animal-based assays. The effects of TDO inhibitors on MDD symptoms were evaluated in a stress-induced depression-like mouse model. In mice, both inhibitors had beneficial effects on stress-induced depressive-like behavioral despair and unhealthy physical status. Moreover, both inhibitors increased the liver serotonin/tryptophan ratio and decreased the kynurenine/tryptophan ratio after oral administration, demonstrating in vivo inhibition of TDO activity. Our data substantiated the potential of TDO inhibition as a therapeutic strategy to improve behavioral activity and decrease despair symptoms in major depressive disorder. CONCLUSIONS: This study introduced a hitherto undocumented comprehensive screening strategy to identify TDO inhibitors in PaeR extract. Our findings also highlighted the potential of PaeR as a source of antidepressant constituents and pinpointed the inhibition of TDO as a promising therapeutic approach for managing major depressive disorder.

The effects of metal cofactors on the reactivity of quercetin 2,4-dioxygenase: synthetic model studies with M(II)-complexes (M = Mn, Co, Ni, Cu, Zn) and assessment of the regulatory factors in catalytic efficacy.

This paper demonstrates the metal ion effects on the quercetin 2,4-dioxygenase (2,4-QD)-like reactivity. For this purpose, a series of five metal(II)-acetato complexes [MII(L)(OAc)] {M = Mn (1OAc), Co (2OAc), Ni (3OAc), Cu (4OAc), Zn (5OAc); OAc = acetate} supported with a newly designed N3O-donor carboxylato ligand L- {L- = 2-((benzyl((6'-methyl-[2,2'-bipyridin]-6-yl)methyl)amino)methyl)benzoate} has been synthesised as models for the active sites of MII-substituted 2,4-QDs. The enzyme-substrate (ES) model complexes [MII(L)(fla)] {M = Mn (1fla), Co (2fla), Ni (3fla), Cu (4fla), Zn (5fla); flaH = flavonol} have been synthesised by reacting flaH with their corresponding acetate-bound complexes in basic conditions. Detailed physicochemical properties of all the compounds are reported. Furthermore, single-crystal X-ray diffractions have been done to determine the structures of the compounds 2OAc·2H2O, 3OAc, 4OAc·CH2Cl2·2H2O, 5OAc·2H2O and 2fla·MeOH. The enzymatic reactivities of complexes 1OAc-5OAc towards the dioxygenation of flavonol have been explored in detail. All the complexes effectively catalyse the oxygenative degradation of flavonol in N,N-dimethylformamide (DMF) medium at 70 °C under multiple-turnover conditions and produce enzyme-type products. Kinetic investigations were performed to see the metal ions' effects on reactivity. The reaction rates vary with the metal ions, showing the order Co > Ni > Zn > Mn > Cu. The studies reveal that the reactivities of the [MII(L)(OAc)] complexes are governed primarily by three factors viz the ES adduct formation constant (Kf), the redox potential (Epa) of the bound fla-/fla˙ couple, and the degree of delocalisation of the fla˙ radical with the metal electrons, which are drastically influenced by the M2+ ions. In the mechanistic interpretation, a single-electron transfer (SET) from the bound-flavonolate to dioxygen has been proposed to generate the catalytically important "M(II)-fla˙" radical and superoxide ion, which react further to bring about the dioxygenation reaction. The identification of the metal(II)-bound flavonoxy radical intermediate for the case of cobalt using EPR spectroscopy and the detection of superoxide ion by NBT2+ test and EPR spin-trapping experiment (DMPO test) are remarkable in envisaging the reaction pathway.

Integration of Molecular Information in Risk Assessment of Patients with Myeloproliferative Neoplasms.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell, characterized by an abnormal proliferation of largely mature cells driven by mutations in JAK2, CALR, and MPL. All these mutations lead to a constitutive activation of the JAK-STAT signaling, which represents a target for therapy. Beyond driver ones, most patients, especially with myelofibrosis, harbor mutations in an array of "myeloid neoplasm-associated" genes that encode for proteins involved in chromatin modification and DNA methylation, RNA splicing, transcription regulation, and oncogenes. These additional mutations often arise in the context of clonal hematopoiesis of indeterminate potential (CHIP). The extensive characterization of the pathologic genome associated with MPN highlighted selected driver and non-driver mutations for their clinical informativeness. First, driver mutations are enlisted in the WHO classification as major diagnostic criteria and may be used for monitoring of residual disease after transplantation and response to treatment. Second, mutation profile can be used, eventually in combination with cytogenetic, histopathologic, hematologic, and clinical variables, to risk stratify patients regarding thrombosis, overall survival, and rate of transformation to secondary leukemia. This review outlines the molecular landscape of MPN and critically interprets current information for their potential impact on patient management.

The potential assessment of green alga Chlamydomonas reinhardtii CC-503 in the biodegradation of benz(a)anthracene and the related mechanism analysis.

Benz(a)anthracene (BaA) is a polycyclic aromatic hydrocarbons (PAHs), that belongs to a group of carcinogenic and mutagenic persistent organic pollutants found in a variety of ecological habitats. In this study, the efficient biodegradation of BaA by a green alga Chlamydomonas reinhardtii (C. reinhardtii) CC-503 was investigated. The results showed that the growth of C. reinhardtii was hardly affected with an initial concentration of 10 mg/L, but was inhibited significantly under higher concentrations of BaA (>30 mg/L) (p < 0.05). We demonstrated that the relatively high concentration of 10 mg/L BaA was degraded completely in 11 days, which indicated that C. reinhardtii had an efficient degradation system. During the degradation, the intermediate metabolites were determined to be isomeric phenanthrene or anthracene, 2,6-diisopropylnaphthalene, 1,3-diisopropylnaphthalene, 1,7-diisopropylnaphthalene, and cyclohexanol. The enzymes involved in the degradation included the homogentisate 1,2-dioxygenase (HGD), the carboxymethylenebutenolidase, the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ubiquinol oxidase. The respective genes encoding these proteins were significantly up-regulated ranging from 3.17 fold to 13.03 fold and the activity of enzymes, such as HGD and Rubisco, was significantly induced up to 4.53 and 1.46 fold (p < 0.05), during the BaA metabolism. This efficient degradation ability suggests that the green alga C. reinhardtii CC-503 may be a sustainable candidate for PAHs remediation.

The Behavior of Trispyrazolylborato-Metal(II)-Flavonolate Complexes as Functional Models for Bacterial Quercetinase-Assessment of the Metal Impact.

A series of five compounds TpMesMFla (TpMes = hydrotris(3-mesityl)pyrazolylborate; M = Mn, Fe, Co, Ni, Zn; Fla = 3-hydroxyflavonolate) has been synthesized as models for the 2,4-quercetin dioxygenase, QueD. The structures have been determined and the complexes proved to be isomorphous. Considering the structures more closely revealed that they differ in the degree of delocalization in the chelate ring formed through the binding of the two O donors of the flavonolate to the metal center, which is also supported by the results of UV-vis and IR spectroscopic investigations. The resulting trend (Zn/Fe > Co > Mn > Ni) is, however, not in line with the one that was found investigating the redox properties of the complexes by cyclic voltammetry (Zn > Fe > Ni > Co > Mn). Notably, from CV clear-cut information could be derived, as the complexes exhibited exceptionally well-behaved quasi-reversible redox transitions, indicating that the Tp ligand stabilizes the flavonolate radical formed in the oxidation process rather well. The fact that the rates, with which the complexes react with O2 in DMF solution, correlate with the position of the flavonolate redox couples, suggest that these reactions proceed via the initial electron transfer from the flavonolate to O2. After the O2 reaction, salicylic acid was identified as one of the products, the formation of which can be explained by the hydrolysis of the depside that should form upon a dioxygenation similar to the QueD enzyme-catalyzed reaction. 18O labeling experiments confirmed the presence of O2 derived O atoms. Mechanistic inferences based on the above results are discussed.

Very-Low-Dose Decitabine Is Effective in Treating Intermediate- or High-Risk Myelodysplastic Syndrome.

Nowadays, the regular recommended dose of decitabine for the treatment of myelodysplastic syndrome (MDS) is 20 mg/m2/day for 5 consecutive days with a relatively high incidence of treatment-related morbidities and costs. In this study, a retrospective and multicenter analysis was performed to explore the very-low-dose decitabine schedule for the treatment of patients with IPSS intermediate- or high-risk MDS. A total of 31 newly diagnosed MDS cases from 14 hospitals in Beijing received decitabine monotherapy (decitabine 6 mg/m2/day intravenously for 7 consecutive days, repeated every 4 weeks). With a medium follow-up of 4 months, 10 patients achieved complete remission (32.3%), 8 (25.8%) partial remission, and 3 (9.7%) hematological improvement. The overall response rate (ORR) was 67.7%. Rates of 21.7% for severe infections and 11.6% for severe bleedings were observed among all courses. The median cost of each course was USD 5,300, 3,000, 2,900, and 2,000, respectively. Multivariate analysis identified bone marrow blast cells ≥10% and a Charlson comorbidity index ≥1 as 2 independent factors for efficacy. In conclusion, very-low-dose decitabine showed relatively good efficacy, good tolerance, and low medical cost in the treatment of intermediate- or high-risk MDS. Elderly patients with more than 1 complication or patients with a higher proportion of blast cells may be the most suitable candidates for this regimen.

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

INTRODUCTION: DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. METHOD: Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). RESULTS: Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. CONCLUSIONS: Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Calorimetric assessment of Fe(2+) binding to α-ketoglutarate/taurine dioxygenase: ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad.

The thermodynamic properties of Fe(2+) binding to the 2-His-1-carboxylate facial triad in α-ketoglutarate/taurine dioxygenase (TauD) were explored using isothermal titration calorimetry. Direct titrations of Fe(2+) into TauD and chelation experiments involving the titration of ethylenediaminetetraacetic acid into Fe(2+)-TauD were performed under an anaerobic environment to yield a binding equilibrium of 2.4 (±0.1) × 10(7) (Kd = 43 nM) and a ΔG° value of -10.1 (±0.03) kcal/mol. Further analysis of the enthalpy/entropy contributions indicates a highly enthalpic binding event, where ΔH = -11.6 (±0.3) kcal/mol. Investigations into the unfavorable entropy term led to the observation of water molecules becoming organized within the Fe(2+)-TauD structure.

Assessment of PET/CT in multifocal myeloid sarcomas with loss of TET2: a case report and literature review.

Myeloid sarcoma is a rare solid tumor consisting of leukemic myeloblasts and/or myeloid precursors occurring outside the blood or bone marrow. The unique site with myeloid sarcoma has been reported, the multiple sites of myeloid sarcoma have rarely been cited in the medical literature. Here we report that the unusual clinical presentation and management of myeloid sarcoma in multiple sites with PET-CT, highlighting the utility of PET-CT was useful in detecting and monitoring myeloid sarcoma. We also found that loss of TET2 and gain of 5 hmC in the case of myeloid sarcoma, indicating the mechanism for myeloid sarcoma is totally different with other hematopoietic malignancies.

Assessment of bacterial bph gene in Amazonian dark earth and their adjacent soils.

Amazonian Anthrosols are known to harbour distinct and highly diverse microbial communities. As most of the current assessments of these communities are based on taxonomic profiles, the functional gene structure of these communities, such as those responsible for key steps in the carbon cycle, mostly remain elusive. To gain insights into the diversity of catabolic genes involved in the degradation of hydrocarbons in anthropogenic horizons, we analysed the bacterial bph gene community structure, composition and abundance using T-RFLP, 454-pyrosequencing and quantitative PCR essays, respectively. Soil samples were collected in two Brazilian Amazon Dark Earth (ADE) sites and at their corresponding non-anthropogenic adjacent soils (ADJ), under two different land use systems, secondary forest (SF) and manioc cultivation (M). Redundancy analysis of T-RFLP data revealed differences in bph gene structure according to both soil type and land use. Chemical properties of ADE soils, such as high organic carbon and organic matter, as well as effective cation exchange capacity and pH, were significantly correlated with the structure of bph communities. Also, the taxonomic affiliation of bph gene sequences revealed the segregation of community composition according to the soil type. Sequences at ADE sites were mostly affiliated to aromatic hydrocarbon degraders belonging to the genera Streptomyces, Sphingomonas, Rhodococcus, Mycobacterium, Conexibacter and Burkholderia. In both land use sites, shannon's diversity indices based on the bph gene data were higher in ADE than ADJ soils. Collectively, our findings provide evidence that specific properties in ADE soils shape the structure and composition of bph communities. These results provide a basis for further investigations focusing on the bio-exploration of novel enzymes with potential use in the biotechnology/biodegradation industry.

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

Assessment of polycyclic aromatic hydrocarbon biodegradation potential in mangrove sediment from Don Hoi Lot, Samut Songkram Province, Thailand.

AIMS: To assess the biodegradation potential of mixed polycyclic aromatic hydrocarbons (PAHs) in mangrove sediments. METHODS AND RESULTS: Sediment microcosms were constructed with sediment collected from Don Hoi Lot, Samut Songkram Province, Thailand, by supplementation with a mixture of acenaphthene, phenanthrene and pyrene. At the end of 8 weeks, low molecular weight PAHs, acenaphthene and phenanthrene were completely degraded. PCR-denaturing gradient gel electrophoresis profile suggests that Marinobacter, Enterobacter and Dethiosulfatibacter play important roles in PAH degradation in mangrove sediment. Furthermore, six PAH-degrading bacteria were isolated consisting of novel phenanthrene-degrading Dyella sp. and Luteibacter sp., phenanthrene-degrading Burkholderia sp., acenaphthene-degrading Alcaligenes sp. and pyrene-degrading Ochrobactrum sp. Moreover, dioxygenase genes could be detected both in sediment microcosms as well as in all of the isolated strains. CONCLUSIONS: These results demonstrated that indigenous bacteria in the mangrove sediment had the ability to degrade phenanthrene in the presence of mixture PAHs with high efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Culture and nonculture methods were combined to assess PAH biodegradation in mangrove sediment. Two novel phenanthrene-degrading bacteria were isolated. Three genera of bacteria that play important roles in PAH degradation were indicated by nonculture approach. Moreover, dioxygenase genes could be detected. This information is useful for further bioremediation of PAH-contaminated mangrove sediments.

Distribution and prediction of catalytic domains in 2-oxoglutarate dependent dioxygenases.

BACKGROUND: The 2-oxoglutarate dependent superfamily is a diverse group of non-haem dioxygenases, and is present in prokaryotes, eukaryotes, and archaea. The enzymes differ in substrate preference and reaction chemistry, a factor that precludes their classification by homology studies and electronic annotation schemes alone. In this work, I propose and explore the rationale of using substrates to classify structurally similar alpha-ketoglutarate dependent enzymes. FINDINGS: Differential catalysis in phylogenetic clades of 2-OG dependent enzymes, is determined by the interactions of a subset of active-site amino acids. Identifying these with existing computational methods is challenging and not feasible for all proteins. A clustering protocol based on validated mechanisms of catalysis of known molecules, in tandem with group specific hidden markov model profiles is able to differentiate and sequester these enzymes. Access to this repository is by a web server that compares user defined unknown sequences to these pre-defined profiles and outputs a list of predicted catalytic domains. The server is free and is accessible at the following URL (http://comp-biol.theacms.in/H2OGpred.html). CONCLUSIONS: The proposed stratification is a novel attempt at classifying and predicting 2-oxoglutarate dependent function. In addition, the server will provide researchers with a tool to compare their data to a comprehensive list of HMM profiles of catalytic domains. This work, will aid efforts by investigators to screen and characterize putative 2-OG dependent sequences. The profile database will be updated at regular intervals.

Structural modeling of heteromeric protein complexes from disassembly pathways and ion mobility-mass spectrometry.

Structure determination of macromolecular protein assemblies remains a challenge for well-established methods. Here, we provide an assessment of an emerging structural technique, ion mobility-mass spectrometry (IM-MS), and examine the use of collision cross-sections (CCSs), derived from IM-MS, as restraints for structure characterization of heteromeric protein assemblies. Using 15 complexes selected from the Protein Data Bank, we validate the use of low-resolution models by comparing their CCSs with those calculated for all-atom structures. We then select six heteromeric complexes, disrupting them in solution to form subcomplexes. Experimental and calculated CCSs reveal close similarity for 18 of the 21 (sub)complexes. Exploring the use of CCS as a restraint, we incorporate it into a scoring function and show good correlation between the score and similarity to the native structure for heteromers, especially when an additional symmetry restraint was introduced.

HMM-FRAME: accurate protein domain classification for metagenomic sequences containing frameshift errors.

BACKGROUND: Protein domain classification is an important step in metagenomic annotation. The state-of-the-art method for protein domain classification is profile HMM-based alignment. However, the relatively high rates of insertions and deletions in homopolymer regions of pyrosequencing reads create frameshifts, causing conventional profile HMM alignment tools to generate alignments with marginal scores. This makes error-containing gene fragments unclassifiable with conventional tools. Thus, there is a need for an accurate domain classification tool that can detect and correct sequencing errors. RESULTS: We introduce HMM-FRAME, a protein domain classification tool based on an augmented Viterbi algorithm that can incorporate error models from different sequencing platforms. HMM-FRAME corrects sequencing errors and classifies putative gene fragments into domain families. It achieved high error detection sensitivity and specificity in a data set with annotated errors. We applied HMM-FRAME in Targeted Metagenomics and a published metagenomic data set. The results showed that our tool can correct frameshifts in error-containing sequences, generate much longer alignments with significantly smaller E-values, and classify more sequences into their native families. CONCLUSIONS: HMM-FRAME provides a complementary protein domain classification tool to conventional profile HMM-based methods for data sets containing frameshifts. Its current implementation is best used for small-scale metagenomic data sets. The source code of HMM-FRAME can be downloaded at http://www.cse.msu.edu/~zhangy72/hmmframe/ and at https://sourceforge.net/projects/hmm-frame/.

Prognostic classification and risk assessment in myelodysplastic syndromes.

The clinical heterogeneity of myelodysplastic syndromes (MDS) is best illustrated by the observation that these disorders range from indolent conditions with a near-normal life expectancy to forms approaching acute myeloid leukemia (AML). A risk-adapted treatment strategy is mandatory for conditions showing a highly variable clinical course, and definition of the individual risk has been based so far on the use of prognostic scoring systems. The authors have developed a prognostic model that accounts for the World Health Organization (WHO) categories, cytogenetics, transfusion dependency, and bone marrow fibrosis. This WHO classification-based Prognostic Scoring System (WPSS) is able to classify patients into five risk groups showing different survivals and probabilities of leukemic evolution. WPSS predicts survival and leukemia progression at any time during follow-up, and may therefore be used for implementing risk-adapted treatment strategies.

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...

Functional gene abundances (nahAc, alkB, xylE) in the assessment of the efficacy of bioremediation.

In this study, we compared the mineralization rates of three selected (14)C-labeled hydrocarbon compounds, octacosane, toluene, and naphthalene, with the presence of the corresponding functional genes (alkB, xylE, nahAc) in a large number of soil samples representing different types of soil and petroleum hydrocarbon contamination. Functional genes were enumerated by the replicate limited dilution (RLD) polymerase chain reaction (PCR) technique. RLD-PCR was further compared to real-time PCR measurements for nahAc and xylE for some samples. At a heating oil-contaminated site, octacosane mineralization rates were higher (on average 0.0015 day(-1)) when compared to aerobic naphthalene and toluene mineralization (on average 0.00003 and 0.0007 day(-1)). The corresponding gene abundances measured by RLD-PCR were on average 0.95, 0.3, and 0.13 x 10(3) gene copies g(-1) soil for alkB, nahAc, and xylE, respectively. At a site contaminated with gasoline, the situation was the opposite: Toluene mineralization was the highest (on average 0.0031 day(-1)), and only xylE genes could be detected (on average 0.13 x 10(3) gene copies g(-1) soil by RLD-PCR). XylE and nahAc gene abundances were correlated with the (14)C-toluene and naphthalene mineralization activities, respectively, in samples from aerobic layers. AlkB gene abundances were not correlated with the octacosane mineralization. Real-time PCR was a more sensitive method than RLD-PCR by a factor of 1,200 for nahAc and 300 for xylE. In conclusion, functional gene abundances seemed to reflect the type of the contamination. With optimized assays, the gene abundances can be used to assess bioremediation efficacy.

Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases.

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic alpha subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant alpha subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase alpha subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an alpha subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.

Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production.

Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIFalpha subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules.