Aldehyde oxidase functions as a superoxide generating NADH oxidase: an important redox regulated pathway of cellular oxygen radical formation

The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress. Utilizing electron paramagnetic resonance spin trapping, we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH and the effects of inhibitors of AO on NADH-mediated superoxide (O(2)(??)) generation. NADH was found to be a good substrate for AO with apparent K(m) and V(max) values of 29 μM and 12 nmol min(-1) mg(-1), respectively. From O(2)(??) generation measurements by cytochrome c reduction the apparent K(m) and V(max) values of NADH for AO were 11 μM and 15 nmol min(-1) mg(-1), respectively. With NADH oxidation by AO, ≥65% of the total electron flux led to O(2)(??) generation. Diphenyleneiodonium completely inhibited AO-mediated O(2)(??) production, confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O(2)(??) generation were studied with amidone the most potent exerting complete inhibition at 100 μM concentration, while 150 μM menadione, raloxifene, or β-estradiol led to 81%, 46%, or 26% inhibition, respectively. From the kinetic data, and the levels of AO and NADH, O(2)(??) production was estimated to be ~89 and ~4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions. Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis.     

Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 microM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12-log10 protections, respectively, at the dose of 50 mg/kg body weight.     

Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.     

Arsenic hyperaccumulation in the Chinese brake fern (Pteris vittata) deters grasshopper (Schistocerca americana) herbivory

Brake fern, Pteris vittata, not only tolerates arsenic but also hyperaccumulates it in the frond. The hypothesis that arsenic hyperaccumulation in this fern could function as a defense against insect herbivory was tested. Fronds from control and arsenic-treated ferns were presented to nymphs of the grasshopper Schistocerca americana. Feeding damage was recorded by visual observation and quantification of the fresh weight of frond left uneaten and number of fecal pellets produced over a 2-d period. Grasshopper weight was determined before and after 5 d of feeding. Grasshoppers consumed significantly greater amounts of the frond tissue, produced more fecal pellets and had increased body weight on control plants compared with grasshoppers fed arsenic-treated ferns. Very little or none of the arsenic-treated ferns were consumed indicating feeding deterrence. In a feeding deterrent experiment with lettuce, sodium arsenite at 1.0 mm deterred grasshoppers from feeding whereas 0.1 mm did not. In a choice experiment, grasshoppers preferred to feed on lettuce dipped in water compared with lettuce dipped in 1.0 mm sodium arsenite. Our results show that arsenic hyperaccumulation in brake fern is an elemental defense against grasshopper herbivory.     

The Usefulness and Feasibility of Mobile Interface in Tuberculosis Notification (MITUN) Voice Based System for Notification of Tuberculosis by Private Medical Practitioners – A Pilot Project

Introduction

Tuberculosis (TB) is a notifiable disease and health care providers are required to notify every TB case to local authorities. We conducted a pilot study to determine the usefulness and feasibility of mobile interface in TB notification (MITUN) voice based system for notification of TB cases by private medical practitioners.

Methodology

The study was conducted during September 2013 to October 2014 in three zones of Chennai, an urban setting in South India. Private clinics wherein services are provided by single private medical practitioners were approached. The steps involved in MITUN included: Registration of the practitioners and notification of TB cases by them through voice interactions. Pre and post-intervention questionnaires were administered to collect information on TB notification practices and feasibility of MITUN after an implementation period of 6 months.

Results

A total of 266 private medical practitioners were approached for the study. Of them, 184 (69%) participated in the study; of whom 11 (6%) practitioners used MITUN for TB notification. Reasons for not using MITUN include lack of time, referral of patients to government facility, issues related to patient confidentiality and technical problems. Suggestions for making mobile phone based TB notification process user-friendly included reducing call duration, including only crucial questions and using missed call or SMS options.

Conclusion

The performance (feasibility and usefulness) of MITUN voice based system for TB notification in the present format was sub-optimal. Perceived problems, logistical and practical issues preclude scale–up of notification of TB by private practitioners.     

Chemoproteomics reveals Toll-like receptor fatty acylation

Background

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.

Results

A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands.

Conclusions

This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

     

αA-Crystallin-Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

Background

A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.

Methodology/Principal Findings

Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.

Conclusion/Significance

These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.     

Coupling microbial catabolic actions with abiotic redox processes: A new recipe for persistent organic pollutant (POP) removal

The continuous release of toxic persistent organic pollutants (POPs) into the environment has raised a need for effective cleanup methods. The tremendous natural diversity of microbial catabolic mechanisms suggests that catabolic routes may be applied to the remediation of POP-contaminated fields. A large number of the recalcitrant xenobiotics have been shown to be removable via the natural catabolic mechanisms of microbes, and detailed biochemical studies of the catabolic methods, together with the development of sophisticated genetic engineering, have led to the use of synthetic microbes for the bioremediation of POPs. However, the steric effects of substituted halogen moieties, microbe toxicity, and the low bioavailability of POPs still deteriorate the efficiency of removal strategies based on natural and synthetic catabolic mechanisms. Recently, abiotic redox processes that induce rapid reductive dehalogenation, hydroxyl radical-based oxidation, or electron shuttling have been reasonably coupled with microbial catabolic actions, thereby compensating for the drawbacks of biotic processes in POP removal. In this review, we first compare the pros and cons of individual methodologies (i.e., the natural and synthetic catabolism of microbes and the abiotic processes involving zero-valent irons, advanced oxidation processes, and small organic stimulants) for POP removal. We then highlight recent trends in coupling the biotic–abiotic methodologies and discuss how the processes are both feasible and superior to individual methodologies for POP cleanup. Cost-effective and environmentally sustainable abiotic redox actions could enhance the microbial bioremediation potential for POPs.