Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

Effects of Disinfectants on Larval Development of Ascaris suum Eggs

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25˚C in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.     

Lab-scale study of an anaerobic membrane bioreactor (AnMBR) for dilute municipal wastewater treatment

Anaerobic bioreactors supplemented with membrane technology have become quite popular, owing to their favorable energy recovery characteristics. In this study, a lab-scale anaerobic Membrane Bioreactor (AnMBR) was assessed in experimental treatments of pre-settled dilute municipal wastewater obtained from a full-scaled wastewater treatment plant. The MBR system was operated in continuous flow mode for 440 days. To evaluate the performance of the AnMBR under various loading rates, the hydraulic retention time (HRT) was reduced in a stepwise manner (from 2 to 0.5 days). Afterward, the mixed liquor suspended solids (MLSS) were reduced from 7,000 to 3,000 mg/L in increments of 1,000 mg/L, resulting in a decrease in solids retention time (SRT) at a constant HRT of 1.0 day. The soluble chemical oxygen demand (SCOD) concentration in the feed varied between 38 and 131 mg/L, whereas the average permeate SCOD ranged between 18 and 37 mg/L, reflecting excellent effluent quality. The AnMBR performance in terms of COD removal proved stable, despite variations in influent characteristics and HRT and SRT changes. The concentration of extracellular polymeric substance (EPS) was reduced with decreases in HRT from 42 to 22 mg VS/mg of MLSS, thereby indicating that the increased biomass concentration biodegraded the EPS at lower HRTs. AnMBR is, therefore, demonstrably a feasible option for the treatment of dilute wastewater with separate stage nitrogen and phosphorus removal processes.     

Allyl Isothiocyanate Ameliorates Angiogenesis and Inflammation in Dextran Sulfate Sodium-Induced Acute Colitis

Allyl isothiocyanate (AITC) is a phytochemical found in cruciferous vegetables that has known chemopreventive and chemotherapeutic activities. Thus far, the antiangiogenic activity of AITC has not been reported in in vivo studies. Herein, we investigated the effect of AITC on angiogenesis and inflammation in a mouse model of colitis. Experimental colitis was induced in mice by administering 3% dextran sulfate sodium via drinking water. To monitor the activity of AITC in this model, we measured body weight, disease activity indices, histopathological scores, microvascular density, myeloperoxidase activity, F4/80 staining, inducible nitric oxide synthase (iNOS) expression, cyclooxygenase-2 (COX-2) expression, and vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) expression in the mice. We found that AITC-treated mice showed less weight loss, fewer clinical signs of colitis, and longer colons than vehicle-treated mice. AITC treatment also significantly lessened the disruption of colonic architecture that is normally associated with colitis and repressed the microvascularization response. Further, AITC treatment reduced both leukocyte recruitment and macrophage infiltration into the inflamed colon, and the mechanism these activities involved repressing iNOS and COX-2 expression. Finally, AITC attenuated the expression of VEGF-A and VEGFR2. Thus, AITC may have potential application in treating conditions marked by inflammatory-driven angiogenesis and mucosal inflammation.