Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway. The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation. To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene. The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT and to other previously described ring cleavage dioxygenases. The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl. However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.     

Differential MR Delayed Enhancement Patterns of Chronic Myocardial Infarction between Extracellular and Intravascular Contrast Media

Objectives

Because the distribution volume and mechanism of extracellular and intravascular MR contrast media differ considerably, the enhancement pattern of chronic myocardial infarction with extracellular or intravascular media might also be different. This study aims to investigate the differences in MR enhancement patterns of chronic myocardial infarction between extracellular and intravascular contrast media.

Materials and Methods

Twenty pigs with myocardial infarction underwent cine MRI, first pass perfusion MRI and delayed enhancement MRI with extracellular or intravascular media at four weeks after coronary occlusion. Myocardial blood flow (MBF) was determined with microsphere measurement. The infarction histopathological changes were evaluated by hematoxylin and eosin staining and Masson''s trichrome method.

Results

Cine MRI revealed the reduced wall thickening in chronic infarction compared with normal myocardium. Moreover, significant wall thinning in chronic infarction was observed in cine MRI. Peak first-pass signal intensity didn’t significantly differ between chronic infarction and normal myocardium no matter what kinds of contrast media. At the following delayed enhancement phase, extracellular media-enhanced signal intensity was significantly higher in chronic infarction than in normal myocardium. Conversely, intravascular media-enhanced signal intensity was almost equivalent among chronic infarction and normal myocardium. At four weeks after infarction, MBF in chronic infarction approached to that in normal myocardium. Large thick-walled vessels were detected at peri-infarction zones. The cardiomyocytes were replaced by scar tissue consisting of dilated blood vessels and discrete fibers of collagen.

Conclusions

Chronic infarction was characterized by the significantly reduced wall thickening and the definite wall thinning. First-pass myocardial perfusion defect was not detected in chronic infarction with two media due to the significantly recovered MBF and well-developed collateral vessels. Infarction remodeling enlarged the extracellular compartment, which was available for extracellular media but not accessible to intravascular media. Extracellular media identified chronic infarction as the hyper-enhancement; nonetheless, intravascular media didn’t provide delayed enhancement.     

Theta,Mental Flexibility,and Post-Traumatic Stress Disorder: Connecting in the Parietal Cortex

Post-traumatic stress disorder (PTSD) is a mental health injury characterised by re-experiencing, avoidance, numbing and hyperarousal. Whilst the aetiology of the disorder is relatively well understood, there is debate about the prevalence of cognitive sequelae that manifest in PTSD. In particular, there are conflicting reports about deficits in executive function and mental flexibility. Even less is known about the neural changes that underlie such deficits. Here, we used magnetoencephalography to study differences in functional connectivity during a mental flexibility task in combat-related PTSD (all males, mean age = 37.4, n = 18) versus a military control (all males, mean age = 33.05, n = 19) group. We observed large-scale increases in theta connectivity in the PTSD group compared to controls. The PTSD group performance was compromised in the more attentionally-demanding task and this was characterised by ''late-stage'' theta hyperconnectivity, concentrated in network connections involving right parietal cortex. Furthermore, we observed significant correlations with the connectivity strength in this region with a number of cognitive-behavioural outcomes, including measures of attention, depression and anxiety. These findings suggest atypical coordination of neural synchronisation in large scale networks contributes to deficits in mental flexibility for PTSD populations in timed, attentionally-demanding tasks, and this propensity toward network hyperconnectivity may play a more general role in the cognitive sequelae evident in this disorder.     

The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.