Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Bioelectricity generation using two chamber microbial fuel cell treating wastewater from food processing

Electricity generation from microbial fuel cells which treat food processing wastewater was investigated in this study. Anaerobic anode and aerobic cathode chambers were separated by a proton exchange membrane in a two-compartment MFC reactor. Buffer solutions and food industry wastewater were used as electrolytes in the anode and cathode chambers, respectively. The produced voltage and current intensity were measured using a digital multimeter. Effluents from the anode compartment were tested for COD, BOD₅, NH₃, P, TSS, VSS, SO₄ and alkalinity. The maximum current density and power production were measured 527 mA/m² and 230 mW/m² in the anode area, respectively, at operation organic loading (OLR) of 0.364 g COD/l.d. At OLR of 0.182 g COD/l.d, maximum voltage and columbic efficiency production were recorded 0.475 V and 21%, respectively. Maximum removal efficiency of COD, BOD₅, NH₃, P, TSS, VSS, SO₄ and alkalinity were 86, 79, 73, 18, 68, 62, 30 and 58%, respectively. The results indicated that catalysts and mediator-less microbial fuel cells (CAML-MFC) can be considered as a better choice for simple and complete energy conversion from the wastewater of such industries and also this could be considered as a new method to offset wastewater treatment plant operating costs.     

Dodonaea viscosa var. angustifolia leaf: New source of polysaccharide and its anti-oxidant activity

Ultrasonic technology was applied for polysaccharide extraction from the leaves of Dodonaea viscosa and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Three independent variables were extraction time (X₁), extraction temperature (X₂) and ultrasonic power (X₃), respectively. The statistical analysis indicated the independent variables (X₁, X₂, X₃), the quadratic terms (X₁₁ and X₃₃) and the interaction terms (X₁X₂, X₁X₃, X₂X₃) had significant effects on the yield of polysaccharides (P < 0.05). The optimal extraction conditions of D. viscosa leaf were determined as follows: extraction time 50.54 min, extraction temperature 85 °C and ultrasonic power 400 W. Under these conditions, the experimental yield of polysaccharides was 9.455 ± 0.24%, which was agreed closely with the predicted value (9.398%). The evaluation of anti-oxidant activity suggested that the polysaccharide exhibited significant protection against DPPH and hydroxyl radicals and could be explored as a nutraceutical agent.     

Parameters Effects Evaluation of Microbial Strengthening of Sandy Soils in Mixing Experiments Using Taguchi Methodology

The effects of experimental parameters including soil type, curing duration, inoculum size, and biomass and nutrients concentration on soil strengthening due to calcite precipitation by Sporosarcina pasteurii PTCC 1645 were investigated. The laboratory-scale mixing experiments on remolded samples were designed by the Taguchi method. Soil type proved to be the most incorporating factor, followed by curing time and nutrient concentration. The main effect and the interactions of the parameters were presented and the optimal conditions were obtained. This suggests the importance of local conditions including soil type on any future large-scale, in situ application.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.     

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon

The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.     

Discovery of highly potent and selective D4 ligands by interactive SAR study

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with K_i 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with K_i (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands’ arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.     

Adipogenetic effects of retrofractamide A derivatives in 3T3-L1 cells

In a previous study, retrofractamide A from the fruit of Piper chaba was shown to promote adipogenesis in 3T3-L1 cells. In the present study, retrofractamide A and its derivatives were synthesized, and their adipogenetic effects in 3T3-L1 cells were examined. Among the tested compounds, an amide composed of 9-(3′,4′-methylenedioxyphenyl)-nona-2E,4E,8E-trienoic acid and an n-butyl or n-pentyl amine showed strongest activity. Moreover, the amide with the n-pentyl amine moiety significantly increased the uptake of 2-deoxyglucose into the cells, and also increased the mRNA levels of adiponectin, peroxisome proliferator-activated receptor γ2 (PPARγ2), glucose transporter 4 (GLUT4), fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein (C/EBP) α and β in a similar manner as the PPARγ agonist troglitazone, although it had less agonistic activity against PPARγ.