Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

Structural basis for the binding of DNP and purine nucleotides onto UCP1.

Uncoupling protein 1 (UCP1) conducts protons through the inner mitochondrial membrane to uncouple mitochondrial respiration from ATP production, thereby converting the electrochemical gradient of protons into heat1,2. The activity of UCP1 is activated by endogenous fatty acids and synthetic small molecules, such as 2,4-dinitrophenol (DNP), and is inhibited by purine nucleotides, such as ATP3-5. However, the mechanism by which UCP1 binds to these ligands remains unknown. Here we present the structures of human UCP1 in the nucleotide-free state, the DNP-bound state and the ATP-bound state. The structures show that the central cavity of UCP1 is open to the cytosolic side. DNP binds inside the cavity, making contact with transmembrane helix 2 (TM2) and TM6. ATP binds in the same cavity and induces conformational changes in TM2, together with the inward bending of TM1, TM4, TM5 and TM6 of UCP1, resulting in a more compact structure of UCP1. The binding site of ATP overlaps with that of DNP, suggesting that ATP competitively blocks the functional engagement of DNP, resulting in the inhibition of the proton-conducting activity of UCP1.

Interaction with 2,4-dinitrophenol correlates with polyreactivity, self-binding, and stability of clinical-stage therapeutic antibodies.

Therapeutic antibodies should cover particular physicochemical and functional requirements for successful entry into clinical practice. Numerous experimental and computational approaches have been developed for early identification of different unfavourable features of antibodies. Immune repertoires of healthy humans contain a fraction of antibodies that recognize nitroarenes. These antibodies have been demonstrated to manifest antigen-binding polyreactivity. Here we observed that >20 % of 112 clinical stage therapeutic antibodies show pronounced binding to 2,4-dinitrophenol conjugated to albumin. This interaction predicts a number of unfavourable functional and physicochemical features of antibodies such as polyreactivity, tendency for self-association, stability and expression yields. Based on these findings we proposed a simple approach that may add to the armamentarium of assays for early identification of developability liabilities of antibodies intended for therapeutic use.

Genome and transcriptome sequencing of a newly isolated 2,4-dinitrophenol-degrading strain Rhodococcus imtechensis XM24D.

BACKGROUND: 2,4-Dinitrophenol (2,4-DNP) is an important organic environmental pollutant that is highly toxic to all forms of living organisms. A gram-positive strain (designated XM24D) was isolated from 2,4-DNP-contaminated soil by an enrichment technique. OBJECTIVE: The study was designed to analyze the ability of XM24D to degrade 2,4-DNP and its analogs and to reveal the degradation pathways of these aromatic compounds. METHODS: The degradation ability of XM24D was tested by a growth experiment. 2,4-DNP and its analog degradation pathways were predicted by genome and comparative transcriptome sequencing. RESULTS: Growth profiles showed that XM24D was able to utilize 2,4-DNP as the sole source of carbon, nitrogen and energy. Analogs of 2,4-DNP, including 4-nitrophenol (PNP) and 2-chloro-4-nitrophenol (2C4NP), can also be degraded by XM24D. Genome analysis showed that the XM24D genome contains two chromosomes with a combined size of 9.08 Mb and an average GC content of 67.07 %. Average nucleotide identity analysis indicated that Rhodococcus imtechensis RKJ300 is the most closely related strain to XM24D. Comparative transcriptome analysis revealed that the 2,4-DNP/PNP/2C4NP degradation pathway in XM24D is highly similar in sequence and organization to the 2,4-DNP degradation pathway in Rhodococcus opacus HL PM-1, the PNP degradation pathway in Rhodococcus opacus SAO101 and the 2C4NP degradation pathway in Rhodococcus imtechensis RKJ300. These results suggested that 2,4-DNP/PNP/2C4NP was degraded via the 2,4-dinitrocyclohexanone/4-nitrocatechol/hydroxyquinol pathway in XM24D. CONCLUSIONS: Genomic and transcriptomic information on XM24D provides a valuable reference for further investigating the evolutionary characteristics of nitrophenol degradation pathways in microorganisms.

Regulation of biohydrogen production by protonophores in novel green microalgae Parachlorella kessleri.

The green microalgae Parachlorella kessleri RA-002 isolated in Armenia can produce biohydrogen (H2) during oxygenic photosynthesis. Addition of protonophores, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNF) enhances H2 yield in P. kessleri. The maximal H2 yield of ~2.20 and 2.08 mmol L-1 was obtained in the presence of 15 µM CCCP and 50 µM DNF, respectively. During dark conditions H2 production by P. kessleri was not observed even in the presence of protonophores, indicating that H2 formation in these algae was mediated by light conditions. The enhancing effect of protonophores can be coupled with dissipation of proton motive force across thylakoid membrane in P. kessleri, facilitating the availability of protons and electrons to [Fe-Fe]-hydrogenase, which led to formation of H2. At the same time H2 production was not observed in the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a specific inhibitor of PS II. Moreover, diuron inhibits H2 yield in P. kessleri in the presence of protonophores. The inhibitory effect of diuron coupled with suppression of electron transfer from PS II. The results showed that in these algae operates PS II-dependent pathway of H2 generation. This study is important for understanding of the mechanisms of H2 production by green microalgae P. kessleri and developing of its biotechnology.

2,4 Dinitrophenol as Medicine.

In the sanctity of pure drug discovery, objective reasoning can become clouded when pursuing ideas that appear unorthodox, but are spot on physiologically. To put this into historical perspective, it was an unorthodox idea in the 1950's to suggest that warfarin, a rat poison, could be repositioned into a breakthrough drug in humans to protect against strokes as a blood thinner. Yet it was approved in 1954 as Coumadin® and has been prescribed to billions of patients as a standard of care. Similarly, no one can forget the horrific effects of thalidomide, prescribed or available without a prescription, as both a sleeping pill and "morning sickness" anti-nausea medication targeting pregnant women in the 1950's. The "thalidomide babies" became the case-in-point for the need of strict guidelines by the U.S. Food & Drug Administration (FDA) or full multi-species teratogenicity testing before drug approval. More recently it was found that thalidomide is useful in graft versus host disease, leprosy and resistant tuberculosis treatment, and as an anti-angiogenesis agent as a breakthrough drug for multiple myeloma (except for pregnant female patients). Decades of diabetes drug discovery research has historically focused on every possible angle, except, the energy-out side of the equation, namely, raising mitochondrial energy expenditure with chemical uncouplers. The idea of "social responsibility" allowed energy-in agents to be explored and the portfolio is robust with medicines of insulin sensitizers, insulin analogues, secretagogues, SGLT2 inhibitors, etc., but not energy-out medicines. The primary reason? It appeared unorthodox, to return to exploring a drug platform used in the 1930s in over 100,000 obese patients used for weight loss. This is over 80-years ago and prior to Dr Peter Mitchell explaining the mechanism of how mitochondrial uncouplers, like 2,4-dinitrophenol (DNP) even worked by three decades later in 1961. Although there is a clear application for metabolic disease, it was not until recently that this platform was explored for its merit at very low, weight-neutral doses, for treating insidious human illnesses and completely unrelated to weight reduction. It is known that mitochondrial uncouplers specifically target the entire organelle's physiology non-genomically. It has been known for years that many neuromuscular and neurodegenerative diseases are associated with overt production of reactive oxygen species (ROSs), a rise in isoprostanes (biomarker of mitochondrial ROSs in urine or blood) and poor calcium (Ca2+) handing. It has also been known that mitochondrial uncouplers lower ROS production and Ca2+ overload. There is evidence that elevation of isoprostanes precedes disease onset, in Alzheimer's Disease (AD). It is also curious, why so many neurodegenerative diseases of known and unknown etiology start at mid-life or later, such as Multiple Sclerosis (MS), Huntington Disease (HD), AD, Parkinson Disease, and Amyotrophic Lateral Sclerosis (ALS). Is there a relationship to a buildup of mutations that are sequestered over time due to ROSs exceeding the rate of repair? If ROS production were managed, could disease onset due to aging be delayed or prevented? Is it possible that most, if not all neurodegenerative diseases are manifested through mitochondrial dysfunction? Although DNP, a historic mitochondrial uncoupler, was used in the 1930s at high doses for obesity in well over 100,000 humans, and so far, it has never been an FDA-approved drug. This review will focus on the application of using DNP, but now, repositioned as a potential disease-modifying drug for a legion of insidious diseases at much lower and paradoxically, weight neutral doses. DNP will be addressed as a treatment for "metabesity", an emerging term related to the global comorbidities associated with the over-nutritional phenotype; obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, cardiovascular disease, but including neurodegenerative disorders and accelerated aging. Some unexpected drug findings will be discussed, such as DNP's induction of neurotrophic growth factors involved in neuronal heath, learning and cognition. For the first time in 80's years, the FDA has granted (to Mitochon Pharmaceutical, Inc., Blue Bell, PA, USA) an open Investigational New Drug (IND) approval to begin rigorous clinical testing of DNP for safety and tolerability, including for the first ever, pharmacokinetic profiling in humans. Successful completion of Phase I clinical trial will open the door to explore the merits of DNP as a possible treatment of people with many truly unmet medical needs, including those suffering from HD, MS, PD, AD, ALS, Duchenne Muscular Dystrophy (DMD), and Traumatic Brain Injury (TBI).

Goethite promoted biodegradation of 2,4-dinitrophenol under nitrate reduction condition.

Iron oxide may interact with other pollutants in the aquatic environments and further influence their toxicity, transport and fate. The current study was conducted to investigate the biodegradation of 2,4-dinitrophenol (2,4-DNP) in the presence of iron oxide of goethite under anoxic condition using nitrate as the electron acceptor. Experiment results showed that the degradation rate of 2,4-DNP was improved by goethite. High performance liquid chromatography-mass spectra analysis results showed that goethite promoted degradation and transformation of 2,4-diaminophenol and 2-amino-4-nitrophenol (2-nitro-4-aminophenol). Microbial community analysis results showed that the abundance of Actinobacteria, which have the potential ability to degrade PAHs, was increased when goethite was available. This might partially explain the higher degradation of 2,4-DNP. Furthermore, another bacterium of Desulfotomaculum reducens which could reduce soluble Fe(III) and nitrate was also increased. Results further confirmed that nanomaterials in the aquatic environment will influence the microbial community and further change the transformation process of toxic pollutants.

Mitochondrial Uncoupler Prodrug of 2,4-Dinitrophenol, MP201, Prevents Neuronal Damage and Preserves Vision in Experimental Optic Neuritis.

The ability of novel mitochondrial uncoupler prodrug of 2,4-dinitrophenol (DNP), MP201, to prevent neuronal damage and preserve visual function in an experimental autoimmune encephalomyelitis (EAE) model of optic neuritis was evaluated. Optic nerve inflammation, demyelination, and axonal loss are prominent features of optic neuritis, an inflammatory optic neuropathy often associated with the central nervous system demyelinating disease multiple sclerosis. Currently, optic neuritis is frequently treated with high-dose corticosteroids, but treatment fails to prevent permanent neuronal damage and associated vision changes that occur as optic neuritis resolves, thus suggesting that additional therapies are required. MP201 administered orally, once per day, attenuated visual dysfunction, preserved retinal ganglion cells (RGCs), and reduced RGC axonal loss and demyelination in the optic nerves of EAE mice, with limited effects on inflammation. The prominent mild mitochondrial uncoupling properties of MP201, with slow elimination of DNP, may contribute to the neuroprotective effect by modulating the entire mitochondria's physiology directly. Results suggest that MP201 is a potential novel treatment for optic neuritis.

Neuroprotective effects of 2,4-dinitrophenol in an acute model of Parkinson's disease.

Neurons depend on mitochondria for homeostasis and survival, and thus, mitochondrial dysfunction has been implicated in neurodegenerative diseases, including Parkinson's disease (PD). Increasing evidence indicates the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), protects neurons against neurodegeneration and enhances neural plasticity. Here, the authors evaluated the protective effects of intraperitoneally (i.p.) administered low dose DNP in an acute mouse model of PD. Mice were administered DNP (1 or 5mg/kg) for 12 consecutive days, and then on day 13, MPTP (20mg/kg, i.p.) was administered four times (with 2h intervals between injections) to induce PD. It was found that MPTP-induced motor dysfunction was ameliorated in the DNP-treated mice versus vehicle-treated controls. Additionally, DNP effectively attenuated dopaminergic neuronal loss observed in MPTP treated mice. Moreover, in primary cultured neurons, DNP at 10µM, but not at 100µM, prevented MPP+-induced cell death and mitochondrial membrane potential (MMP) reduction. In addition, DNP was observed to cause the nuclear translocation of Nrf2 in primary neurons. Taken together, these findings of the present study suggest that DNP protects dopaminergic neurons against neurodegeneration and maintains MMP integrity in PD by activating adaptive stress responses.

Effect of oxidation and catalytic reduction of trace organic contaminants on their activated carbon adsorption.

The combination of ozonation and activated carbon (AC) adsorption is an established technology for removal of trace organic contaminants (TrOCs). In contrast to oxidation, reduction of TrOCs has recently gained attention as well, however less attention has gone to the combination of reduction with AC adsorption. In addition, no literature has compared the removal behavior of reduction vs. ozonation by-products by AC. In this study, the effect of pre-ozonation vs pre-catalytic reduction on the AC adsorption efficiency of five TrOCs and their by-products was compared. All compounds were susceptible to oxidation and reduction, however the catalytic reductive treatment proved to be a slower reaction than ozonation. New oxidation products were identified for dinoseb and new reduction products were identified for carbamazepine, bromoxynil and dinoseb. In terms of compatibility with AC adsorption, the influence of the oxidative and reductive pretreatments proved to be compound dependent. Oxidation products of bromoxynil and diatrizoic acid adsorbed better than their parent TrOCs, but oxidation products of atrazine, carbamazepine and dinoseb showed a decreased adsorption. The reductive pre-treatment showed an enhanced AC adsorption for dinoseb and a major enhancement for diatrizoic acid. For atrazine and bromoxynil, no clear influence on adsorption was noted, while for carbamazepine, the reductive pretreatment resulted in a decreased AC affinity. It may thus be concluded that when targeting mixtures of TrOCs, a trade-off will undoubtedly have to be made towards overall reactivity and removal of the different constituents, since no single treatment proves to be superior to the other.

Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.

Reactive oxygen species (ROS) are one of the main causes for decreased viability in cryopreserved sperm. Many studies have reported the beneficial effect of antioxidant supplements in freezing media for post-thaw sperm quality. In the present study, we explored two new approaches of ROS inhibition in sperm cryopreservation of yellow catfish, namely mitochondrial-targeted antioxidant and metabolic modulator targeting mitochondrial uncoupling pathways. Our study revealed that addition of MitoQ, a compound designed to deliver ubiquinone into mitochondria, significantly decreased ROS production, as well as lipid peroxidation, and increased post-thaw viability. Similarly, sperm incubated with 2,4-dinitrophenol (DNP), a chemical protonophore that induces mitochondrial uncoupling, also had reduced ROS production, as well as lipid peroxidation, and increased post-thaw sperm viability. Conversely, activation of uncoupling protein (UCP2) by 4-hydroxynonenal (HNE) neither reduced ROS production nor increased post-thaw sperm viability. Our findings indicate that ROS inhibition through mitochondrial-targeted antioxidant or mild mitochondrial uncoupling is beneficial for sperm cryopreservation in yellow catfish. Our study provides novel methods to mitigate oxidative stress induced damage in cryopreserved sperm for future applications.

Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.

MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined 7 point mutants generated by random mutagenesis and 11 site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the α1 and α2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR.

A prototypical small-molecule modulator uncouples mitochondria in response to endogenous hydrogen peroxide production.

A high membrane potential across the mitochondrial inner membrane leads to the production of the reactive oxygen species (ROS) implicated in aging and age-related diseases. A prototypical drug for the correction of this type of mitochondrial dysfunction is presented. MitoDNP-SUM accumulates in mitochondria in response to the membrane potential due to its mitochondria-targeting alkyltriphenylphosphonium (TPP) cation and is uncaged by endogenous hydrogen peroxide to release the mitochondrial uncoupler, 2,4-dinitrophenol (DNP). DNP is known to reduce the high membrane potential responsible for the production of ROS. The approach potentially represents a general method for the delivery of drugs to the mitochondrial matrix through mitochondria targeting and H(2)O(2)-induced uncaging.

Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains.

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.

Accelerated biodegradation of nitrophenols in the rhizosphere of Spirodela polyrrhiza.

We investigated the biodegradation of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP) in the rhizosphere of Spirodela polyrrhiza plants by conducting degradation experiments with three river water samples supplemented with each nitrophenol (NP). We then isolated NP-degrading bacteria both from the S. polyrrhiza roots and from the river water. In the river water samples, removal of the three NP was accelerated in the presence of S. polyrrhiza plants. The three NPs persisted in an autoclaved solution with sterile plants suggests that NP removal was accelerated largely by bacterial NP biodegradation rather than by adsorption and uptake by the plants. We isolated 8 strains of NP-degrading bacteria: 6 strains from the S. polyrrhiza roots and 2 strains from river water without the plants. The 2-NP- and 2,4-DNP-degrading bacteria were isolated only from the S. polyrrhiza roots. The 4-NP-degrading bacteria different from those isolated from the river water samples were also found on S. polyrrhiza roots. The 2-NP- and 4-NP-degrading strains isolated from the roots utilized the corresponding NP (0.5 mmol/L) as the sole carbon and energy source. The 2,4-DNP-degrading strains isolated from the roots showed substantial 2,4-DNP-degrading activity, but the presence of other carbon and energy sources was required for their growth. The isolated NP-degrading bacteria from the roots must have contributed to the accelerated degradation of the three NPs in the rhizosphere of S. polyrrhiza. Our results suggested that rhizoremediation with S. polyrrhiza may be effective for NP-contaminated surface water.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes.

Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (Rana temporaria) tadpoles.

Mitochondria are known to play a central role in life history processes, being the main source of reactive oxygen species (ROS), which promote oxidative constraint. Surprisingly, although the main role of the mitochondria is to produce ATP, the plasticity of mitochondrial ATP generation has received little attention in life history studies. Yet, mitochondrial energy transduction represents the physiological link between environmental resources and energy allocated to animal performance. Studying both facets of mitochondrial functioning (ATP and ROS production) would allow better understanding of the proximate mechanisms underlying life history. We have experimentally modulated the mitochondrial capacity to generate ROS and ATP during larval development of Rana temporaria tadpoles, via chronic exposure (34 days) to a mitochondrial uncoupler (2,4-dinitrophenol, dNP). The aim was to better understand the impact of mitochondrial uncoupling on both responses in terms of oxidative balance, energy input (oxygen and feeding consumption) and energy output (growth and development of the tadpole). Exposure to 2,4-dNP reduced mitochondrial ROS generation, total antioxidant defences and oxidative damage in treated tadpoles compared with controls. Despite the beneficial effect of dNP on oxidative status, development and growth rates of treated tadpoles were lower than those in the control group. Treatment of tadpoles with 2,4-dNP promoted a mild mitochondrial uncoupling and enhanced metabolic rate. These tadpoles did not increase their food consumption, and thus failed to compensate for the energy loss elicited by the decrease in the efficiency of ATP production. These data suggest that the cost of ATP production, rather than the oxidative balance, is the parameter that constrains growth/development of tadpoles, highlighting the central role of energy transduction in larval performance.

Mechanisms of efficient arsenite uptake by arsenic hyperaccumulator Pteris vittata.

Arsenate (AsV) and arsenite (AsIII) are two dominant arsenic species in the environment. While arsenate uptake is via phosphate transporter in plants, including arsenic hyperaccumulator Pteris vittata , AsIII uptake mechanisms by P. vittata are unclear. In this study, we investigated AsIII uptake by P. vittata involving root radial transport from external medium to cortical cells and xylem loading. In the root symplastic solution, AsIII was the predominant species (90-94%) and its concentrations were 1.6-21 times those in the medium. AsIII influx into root symplast followed Michaelis-Menten kinetics with K(m) of 77.7 µM at external AsIII concentrations of 2.6-650 µM. In the presence of metabolic inhibitor 2,4-dinitrophenol (DNP), arsenic concentrations in the root symplast were reduced to the levels lower than in the medium, indicating that a transporter-mediated active process was mainly responsible for AsIII influx into P. vittata roots. Unlike radial transport, AsIII loading into xylem involved both high- and low-affinity systems with K(m) of 8.8 µM and 70.4 µM, respectively. As indicated by the effect of 2,4-DNP, passive diffusion became more important in arsenic loading into xylem at higher external AsIII. The unique AsIII uptake system in P. vittata makes it a valuable model to understand the mechanisms of arsenic hyperaccumulation in the plant kingdom.

Voltammetric and amperometric determination of 2,4-dinitrophenol metabolites.

Methods for determination of 2-amino-4-nitrophenol and 4-amino-2-nitrophenol, metabolites of 2,4-dinitrophenol, were developed using differential pulse (DP) voltammetry and HPLC with amperometric and spectrophotometric detection. The applicability of these methods was tested by the determination of the analytes in model samples of urine after preliminary separation by solid-phase extraction. Voltammetry enabled parallel determination of both analytes, but its application in real matrix was severely limited due to the interference of other compounds present in urine. HPLC allowed the determination in real urine matrix down to micromolar concentrations; amperometric detection proved to be more sensitive and selective than the spectrophotometric one.