Practical NIR Assay Derived from Cyanine to Evaluate Intracellular H2S in Living Cell Imaging.

To monitor the biological function of H2S in real time, this investigation demonstrated the design and synthesis of a novel fluorescent probe integrated with cyanine and 2,4-dinitrophenol for the qualitative and quantitative detection of H2S. An NIR sensitive sensor (FS-HS-1) was provided with a straightforward process. Spectroscopy experiments elucidated that FS-HS-1 could selectively detect H2S in a PBS solution (containing 40% acetonitrile) with a 111-fold fluorescence enhancement at 715 nm (ex. 605 nm). The response towards NaHS occurred in less than 2 min, and the detection limit was confirmed to be as low as 4.47 ± 0.11 nmol/L. Furthermore, the probe is capable of monitoring changes in exogenous H2S concentrations within living cells with confocal and 2P imaging.

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.

Study on the biodegradability improvement of 2,4 dinitrophenol in wastewater using advanced oxidation/reduction process with UV/SO3/ZnO.

2,4-Dinitrophenol (2,4-DNP) is a toxic compound that is widely used in many industrial and agricultural processes. This compound has low biodegradability in the environment due to its aromatic structure, and it is unsuccessfully eliminated by other chemical methods. Therefore, in this study, an integrated oxidation and reduction method was used to remove 2,4-DNP from the aqueous medium, in order to simultaneously use the benefits of oxidizing and reducing radicals in 2,4-DNP degradation. 2,4-DNP degradation was modeled by response surface methodology (RSM) and central composite design (CCD). According to the results obtained from RSM, the optimal values for the studied parameters were obtained at pH = 8.9, time = 25 min, ZnO dose = 0.78 g/L, SO3 = 1.89 mmolL-1 and 2,4-DNP concentration = 5 mg/L. Also, the removal efficiency with the integrated process was 3 to 4 times higher than the advanced oxidation or advanced reduction processes alone. Analysis of the data showed that at the time of the study, 2,4-DNP had been converted to linear hydrocarbons, and increased periods of time were required for complete mineralization. A decrease in the first-order model rate constant (kobs) and an increase in 2,4-DNP degradation rate (robs) were observed at higher DNP concentrations.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

A Comparative Study of the Action of Protonophore Uncouplers and Decoupling Agents as Inducers of Free Respiration in Mitochondria in States 3 and 4: Theoretical and Experimental Approaches.

Theoretical and experimental studies have revealed that that in the liver mitochondria an increase in the rate of free respiration in state 3 induced by protonophore uncouplers 2,4-dinitrophenol and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone is equal to or slightly greater than the increase in respiration rate in state 4 induced by these uncouplers. In contrast to these protonophore uncouplers, the decoupler α,ω-tetradecanedioic acid, increasing the rate of respiration in state 4, does not significantly affect the rate of free respiration in state 3. We have proposed quantitative indicators that allow determining the constituent part of the rate of respiration in state 4, associated with the decoupling effect of the uncoupler. Using the example of palmitic acid, we have found out the fundamental possibility of the simultaneous functioning of uncouplers by two mechanisms: as protonophores and as decouplers. The data obtained contradict the delocalized version of Mitchell's chemiosmotic theory, but are in complete agreement with its local version. It can be assumed that the F0F1-ATP synthase and nearby respiratory chain complexes form a local zone of coupled respiration and oxidative ATP synthesis (zones of oxidative phosphorylation). The uncoupler-induced stimulation of mitochondrial free respiration of mitochondria in state 3 is mainly due to the return of protons to the matrix in local zones, where the generation of a proton motive force (Δр) by respiratory chain complexes is associated with various transport processes, but not with ATP synthesis (zones of protonophore uncoupling). In contrast, respiratory stimulation in state 4 by decouplers is realized in local zones of oxidative phosphorylation by switching the respiratory chain complexes to the idle mode of operation in the absence of ATP synthesis.

Usnic acid as calcium ionophore and mast cells stimulator.

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity.

A growing class of immunotherapeutics work by redirecting components of the immune system to recognize markers on the surface of cancer cells. However, such modalities will remain confined to a relatively small subgroup of patients because of the lack of universal targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidity of solid tumors to selectively graft cancer cells with immuno-engager epitopes. Our targeting approach is based on pHLIP, a unique peptide that selectively targets tumors in vivo by anchoring to cancer cell surfaces in a pH-dependent manner. We established that pHLIP-antigen conjugates trigger the recruitment of antibodies to the surface of cancer cells and induce cytotoxicity by peripheral blood mononuclear and engineered NK cells. These results indicate that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent problems associated with narrow windows of selectivity.

Direct readout protonophore induced selective uncoupling and dysfunction of individual mitochondria within cancer cells.

Concurrently, manipulation of mitochondrial activity and its monitoring have enormous significance in cancer therapy and diagnosis. In this context, a fluorescent probe MitoDP has been developed for validating H2S mediated protonophore (2,4-dinitrophenol, DNP) induced mitochondrial membrane potential change, ROS formation and ATP depletion in cancer cells. The extent of protonophore activation for mitochondrial dysfunction is monitored through fluorescence signalling at 450 nm. The current study provides a proof for the concept of endogenous H2S-mediated controlled and spatial release of bioactive agents, or toxins specifically in mitochondria of cancer cells.

Kinetics of Glutathione Depletion and Antioxidant Gene Expression as Indicators of Chemical Modes of Action Assessed in Vitro in Mouse Hepatocytes with Enhanced Glutathione Synthesis.

Here we report a vertically integrated in vitro - in silico study that aims to elucidate the molecular initiating events involved in the induction of oxidative stress (OS) by seven diverse chemicals (cumene hydroperoxide, t-butyl hydroperoxide, hydroquinone, t-butyl hydroquinone, bisphenol A, Dinoseb, and perfluorooctanoic acid). To that end, we probe the relationship between chemical properties, cell viability, glutathione (GSH) depletion, and antioxidant gene expression. Concentration-dependent effects on cell viability were assessed by MTT assay in two Hepa-1 derived mouse liver cell lines: a control plasmid vector transfected cell line (Hepa-V), and a cell line with increased glutamate-cysteine ligase (GCL) activity and GSH content (CR17). Changes to intracellular GSH content and mRNA expression levels for the Nrf2-driven antioxidant genes Gclc, Gclm, heme oxygenase-1 ( Hmox1), and NADPH quinone oxidoreductase-1 ( Nqo1) were monitored after sublethal exposure to the chemicals. In silico models of covalent and redox reactivity were used to rationalize differences in activity of quinones and peroxides. Our findings show CR17 cells were generally more resistant to chemical toxicity and showed markedly attenuated induction of OS biomarkers; however, differences in viability effects between the two cell lines were not the same for all chemicals. The results highlight the vital role of GSH in protecting against oxidative stress-inducing chemicals as well as the importance of probing molecular initiating events in order to identify chemicals with lower potential to cause oxidative stress.

Substrate-Induced Self-Assembly of Cooperative Catalysts.

Dissipative self-assembly processes in nature rely on chemical fuels that activate proteins for assembly through the formation of a noncovalent complex. The catalytic activity of the assemblies causes fuel degradation, resulting in the formation of an assembly in a high-energy, out-of-equilibrium state. Herein, we apply this concept to a synthetic system and demonstrate that a substrate can induce the formation of vesicular assemblies, which act as cooperative catalysts for cleavage of the same substrate.

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.

Synthesis and characterization of FeIII(µ-OH)ZnII complexes: effects of a second coordination sphere and increase in the chelate ring size on the hydrolysis of a phosphate diester and DNA.

The synthesis and characterization of three ligands and their respective heterobinuclear FeIIIZnII complexes were carried out, with the goal of mimicking the active site of purple acid phosphatases (PAPs). The ligand 2-hydroxy-3-(((2-hydroxy-5-methyl-3-(((2-(pyridin-2-yl)ethyl)(pyridin-2-ylmethyl)amino)methyl)benzyl)(pyridin-2ylmethyl)amino)methyl)-5-methylbenzaldehyde (H2L2) was synthesized and its complex (FeIIIZnIIL2) was used as a basis for comparison with similar complexes previously published in the literature. Subsequent modifications were conducted in the aldehyde group, where 1,2-ethanediamine and 1,4-diaminobutane were used as side chain derivatives. The compounds FeIIIZnIIL2 (1), FeIIIZnIIL2-et (2) and FeIIIZnIIL2-but (3) were characterized by spectroscopic methods (infrared and UV-Vis) and ESI-MS spectrometry. Theoretical calculations were performed to provide insights into the complex structures with FeIIIZnII structures. The hydrolytic activity was analyzed both with the model substrate 2,4-BDNPP and with DNA catalyzed by complexes 1, 2 and 3.

Distribution, lipid-bilayer affinity and kinetics of the metabolic effects of dinoseb in the liver.

Dinoseb is a highly toxic pesticide of the dinitrophenol group. Its use has been restricted, but it can still be found in soils and waters in addition to being a component of related pesticides that, after ingestion by humans or animals, can originate the compound by enzymatic hydrolysis. As most dinitrophenols, dinoseb uncouples oxidative phosphorylation. In this study, distribution, lipid bilayer affinity and kinetics of the metabolic effects of dinoseb were investigated, using mainly the isolated perfused rat liver, but also isolated mitochondria and molecular dynamics simulations. Dinoseb presented high affinity for the hydrophobic region of the lipid bilayers, with a partition coefficient of 3.75×104 between the hydrophobic and hydrophilic phases. Due to this high affinity for the cellular membranes dinoseb underwent flow-limited distribution in the liver. Transformation was slow but uptake into the liver space was very pronounced. For an extracellular concentration of 10µM, the equilibrium intracellular concentration was equal to 438.7µM. In general dinoseb stimulated catabolism and inhibited anabolism. Half-maximal stimulation of oxygen uptake in the whole liver occurred at concentrations (2.8-5.8µM) at least ten times above those in isolated mitochondria (0.28µM). Gluconeogenesis and ureagenesis were half-maximally inhibited at concentrations between 3.04 and 5.97µM. The ATP levels were diminished, but differently in livers from fed and fasted rats. Dinoseb disrupts metabolism in a complex way at concentrations well above its uncoupling action in isolated mitochondria, but still at concentrations that are low enough to be dangerous to animals and humans even at sub-lethal doses.

Study of the interaction between DNP and DIDS with human hemoglobin as binary and ternary systems: spectroscopic and molecular modeling investigation.

The combination of several drugs is necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease in the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. Here, the interaction between 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and 2,4-Dinitrophenol (DNP) with Hemoglobin (Hb) was investigated by different spectroscopic and molecular modeling techniques. Fluorescence analysis was used to estimate the effect of the DIDS and DNP on Hb as well as to define the binding properties of binary and ternary complexes. The distance r between donor and acceptor was obtained by the FRET and found to be 2.25 and 2.13 nm for DIDS and DNP in binary and 2.08 and 2.07 nm for (Hb-DNP) DIDS and (Hb-DIDS) DNP complexes in ternary systems, respectively. Time-resolved fluorescence spectroscopy confirmed static quenching for Hb in the presence of DIDS and DNP in both systems. Furthermore, an increase in ellipticity values of Hb upon interaction with DIDS and DNP showed secondary structural changes of protein that determine to disrupt of hydrogen bonds and electrostatic interactions. Our results showed that the Hb destabilize in the presence of DIDS and DNP. Molecular modeling of the possible binding sites of DIDS and DNP in binary and ternary systems in Hb confirmed the experimental results.

[Fatal course following poisoning with an illegal weight-reducing agent]. / Fatalt forløb efter forgiftning med illegalt slankemiddell.

DNP is a weight-reducing agent, which has been revived through sale over the Internet. DNP uncouples the oxidative phosphorylation in cells, leading to an excessive production of heat. A 39-year-old male ingested four grams of DNP and developed severe muscular stiffness and eventually cardiac arrest. Intubation was unsuccessful, and tracheotomy was performed on scene. Ventilation proved impossible, and the patient was declared dead in the pre-hospital setting. Doctors need to recognize potential lethal intoxications. Symptomatic treatment is warranted.

Reactivity of Imidazole Derivatives toward Phosphate Triester in DMSO/Water Mixtures: A Comprehensive Study on the Solvent Effect.

Many imidazole (IMZ) derivatives of pharmaceutical interest, which are potentially catalytic in dephosphorylation reactions, are soluble solely in mixtures of water and organic solvent. In order to understand these poorly explored reactions and properly compare them, a thorough study related to solvent effects for the analogous spontaneous reaction and with common IMZ derivatives is necessary, which is lacking in the literature. Herein, we report a quantitative solvent effect analysis in DMSO/water mixtures for (i) the hydrolysis reaction of diethyl 2,4-dinitrophenylphosphate (DEDNPP) and (ii) the nucleophilic reaction of IMZ and 1-methylimidazole (MEI) with DEDNPP. The solvent effect was fitted satisfactorily with multiple regression analysis, correlating the obtained second-order rate constants with solvent parameters such as acidity, basicity, and polarity/polarizability from Catalán's scale. The contribution of these parameters can be taken into account to elucidate the reactivity in these media. Interestingly, IMZ is more reactive than MEI in DMSO, compared to water alone, which is attributed to the availability of hydrogen-bond formation. Nuclear magnetic resonance spectroscopy ((1)H, (13)C, and (31)P), mass spectrometry, thermodynamic analysis, and density functional theory calculations were carried out to corroborate the proposed nucleophilic mechanism.

Phosphoester hydrolysis: the incoming substrate turns the bridging hydroxido nucleophile into a terminal one.

Identifying the active nucleophile in hydrolysis reactions catalyzed by binuclear hydrolases is a recurrent problem and a matter of intense debate. We report on the phosphate ester hydrolysis by a Fe(III)Fe(II) complex of a binucleating ligand. This complex presents activities in the range of those observed for similar biomimetic compounds in the literature. The specific electronic properties of the Fe(III)Fe(II) complex allowed us to use (1)H NMR and Mössbauer spectroscopies to investigate the nature of the various species present in the solution in the pH range of 5-10. Both techniques showed that the hydrolysis activity is associated to a µ-hydroxido Fe(III)Fe(II) species. Further (1)H NMR experiments show that binding of anions or the substrate changes this bonding mode suggesting that a terminal hydroxide is the likely nucleophile in these hydrolysis reactions. This view is further supported by the structure determination of the hydrolysis product.

Environmental fate of 2,4-dinitroanisole (DNAN) and its reduced products.

Several defense departments intend to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations by the less sensitive 2,4-dinitroanisole (DNAN). To help understand environmental behavior and ecological risk associated with DNAN we investigated its key initial abiotic and biotic reaction routes and determined relevant physicochemical parameters (pKa, logKow, aqueous solubility (Sw), partition coefficient (Kd)) for the chemical and its products. Reduction of DNAN with either zero valent iron or bacteria regioselectively produced 2-amino-4-nitroanisole (2-ANAN) which, under strict anaerobic conditions, gave 2,4-diaminoanisole (DAAN). Hydrolysis under environmental conditions was insignificant whereas photolysis gave photodegradable intermediates 2-hydroxy-4-nitroanisole and 2,4-dinitrophenol. Physicochemical properties of DNAN and its amino products drastically depended on the type and position of substituent(s) on the aromatic ring. Sw followed the order (TNT

Dinitrophenol modulates gene expression levels of angiogenic, cell survival and cardiomyogenic factors in bone marrow derived mesenchymal stem cells.

Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for better regeneration.

A NEW SPECTROPHOTOMETRIC METHOD FOR THE ASSAY OF LISINOPRIL.

AIM: A new spectrophotometric method for the assay of lisinopril using 2,4-dinitrophenol as reagent is described. MATERIAL AND METHODS: The method involved the addition of 2,4-dinitrophenol to lisinopril in methanol followed by absorbance measurement at 400 nm. Experimental conditions that provide wide linear range, maximum sensitivity, selectivity, accuracy and precisions were optimized. RESULTS AND DISCUSSIONS: Beer's law was obeyed in the concentration range 2.0-14.0 µg/mL. Linear regression equation of calibration graph was A = 0.005 + 0.045 x concentration, with a regression coefficient (r) of 0.9995 (n = 8). The limits of detection (LOD) and quantification (LOQ) calculated according to the ICH guidelines were 0.42 and 1.42 µg/mL, respectively. Accuracy and precision of the assays were determined by computing the intra-day and inter-day variations at three different lisinopril concentrations; the intra-day and inter-day RSD was < 1.43% and accuracy was better than 1.72%. CONCLUSIONS: The proposed method is simple, easy to perform, sensitive, linear, precise, accurate and robust.