Application of Hydrogen Peroxide to the Control of Eutrophic Lake Systems in Laboratory Assays

We exposed water samples from a recreational lake dominated by the cyanobacterium Planktothrix agardhii to different concentrations of hydrogen peroxide (H₂O₂). An addition of 0.33 mg·L⁻¹ of H₂O₂ was the lowest effective dose for the decay of chlorophyll-a concentration to half of the original in 14 h with light and 17 h in experiments without light. With 3.33 mg·L⁻¹ of H₂O₂, the values of the chemical oxygen demand (COD) decreased to half at 36 and 126 h in experiments performed with and without light, respectively. With increasing H₂O₂, there is a decrease in the total and faecal coliform, and this effect was made more pronounced by light. Total and faecal coliform were inhibited completely 48 h after addition of 3.33 mg·L⁻¹ H₂O₂. Although the densities of cyanobacterial cells exposed to H₂O₂ did not decrease, transmission electron microscope observation of the trichomes showed several stages of degeneration, and the cells were collapsed after 48 h of 3.33 mg·L⁻¹ of H₂O₂ addition in the presence of light. Our results demonstrate that H₂O₂ could be potentially used in hypertrophic systems because it not only collapses cyanobacterial cells and coliform bacteria but may also reduce chlorophyll-a content and chemical oxygen demand.     

Effects of Hydrogen Peroxide on Different Toxigenic and Atoxigenic Isolates of Aspergillus flavus

Drought stress in the field has been shown to exacerbate aflatoxin contamination of maize and peanut. Drought and heat stress also produce reactive oxygen species (ROS) in plant tissues. Given the potential correlation between ROS and exacerbated aflatoxin production under drought and heat stress, the objectives of this study were to examine the effects of hydrogen peroxide (H₂O₂)-induced oxidative stress on the growth of different toxigenic (+) and atoxigenic (−) isolates of Aspergillus flavus and to test whether aflatoxin production affects the H₂O₂ concentrations that the isolates could survive. Ten isolates were tested: NRRL3357 (+), A9 (+), AF13 (+), Tox4 (+), A1 (−), K49 (−), K54A (−), AF36 (−), and Aflaguard (−); and one A. parasiticus isolate, NRRL2999 (+). These isolates were cultured under a H₂O₂ gradient ranging from 0 to 50 mM in two different media, aflatoxin-conducive yeast extract-sucrose (YES) and non-conducive yeast extract-peptone (YEP). Fungal growth was inhibited at a high H₂O₂ concentration, but specific isolates grew well at different H₂O₂ concentrations. Generally the toxigenic isolates tolerated higher concentrations than did atoxigenic isolates. Increasing H₂O₂ concentrations in the media resulted in elevated aflatoxin production in toxigenic isolates. In YEP media, the higher concentration of peptone (15%) partially inactivated the H₂O₂ in the media. In the 1% peptone media, YEP did not affect the H₂O₂ concentrations that the isolates could survive in comparison with YES media, without aflatoxin production. It is interesting to note that the commercial biocontrol isolates, AF36 (−), and Aflaguard (−), survived at higher levels of stress than other atoxigenic isolates, suggesting that this testing method could potentially be of use in the selection of biocontrol isolates. Further studies will be needed to investigate the mechanisms behind the variability among isolates with regard to their degree of oxidative stress tolerance and the role of aflatoxin production.     

Pt-MWCNT modified carbon electrode strip for rapid and quantitative detection of H2O2 in food

A single-use screen-printed carbon electrode strip was designed and fabricated. Nanohybrids, prepared by deposition of platinum (Pt) nanoparticles on multi-wall carbon nanotube (MWCNT), was modified on the surface of screen-printed carbon electrode for the development of a fast, sensitive and cost-effective hydrogen peroxide (H₂O₂) detection amperometric sensor strip. With Pt-MWCNT nanohybrids surface modification, current generated in response to H₂O₂ by the screen-printed carbon electrode strip was enhanced 100 fold with an applied potential of 300 mV. Quality of as-prepared electrode strip was assured by the low coefficient of variation (CV) (<5%) of currents measured at 5 s. Three linear detection ranges with sensitivity of 75.2, 120.7, and 142.8 μA mM^?1 cm^?2 were observed for H₂O₂ concentration in the range of 1–15 mM, 0.1–1 mM, and 10–100 μM, respectively. The lowest H₂O₂ concentration could be measured by the as-prepared strip was 10 μM. H₂O₂ levels in green tea infusion and pressed Tofu could be rapidly detected with results comparable to that measured by ferrous oxidation xylenol orange (FOX) assay and peroxidase colorimetric method.     

Oxidation of Cylindrospermopsin by Fenton Process: A Bench-Scale Study of the Effects of Dose and Ratio of H2O2 and Fe(II) and Kinetics

The cyanotoxin cylindrospermopsin (CYN) has become a significant environmental and human health concern due to its high toxicological potential and widespread distribution. High concentrations of cyanotoxins may be produced during cyanobacterial blooms. Special attention is required when these blooms occur in sources of water intended for human consumption since extracellular cyanotoxins are not effectively removed by conventional water treatments, leading to the need for advanced water treatment technologies such as the Fenton process to produce safe water. Thus, the present study aimed to investigate the application of the Fenton process for the degradation of CYN at bench-scale. The oxidation of CYN was evaluated by Fenton reaction at H₂O₂/Fe(II) molar ratio in a range of 0.4 to 4.0, with the highest degradation of about 81% at molar ratio of 0.4. Doubling the concentrations of reactants for the optimized H₂O₂/Fe(II) molar ratio, the CYN degradation efficiency reached 91%. Under the conditions studied, CYN degradation by the Fenton process followed a pseudo-first-order kinetic model with an apparent constant rate ranging from 0.813 × 10⁻³ to 1.879 × 10⁻³ s⁻¹.     

Histamine-induced vasodilatation in the human forearm vasculature

Aim

To investigate the mechanism of action of intra-arterial histamine in the human forearm vasculature.

Methods

Three studies were conducted to assess changes in forearm blood flow (FBF) using venous occlusion plethysmography in response to intra-brachial histamine. First, the dose–response was investigated by assessing FBF throughout a dose-escalating histamine infusion. Next, histamine was infused at a constant dose to assess acute tolerance. Finally, a four way, double-blind, randomized, placebo-controlled crossover study was conducted to assess FBF response to histamine in the presence of H₁- and H₂-receptor antagonists. Flare and itch were assessed in all studies.

Results

Histamine caused a dose-dependent increase in FBF, greatest with the highest dose (30 nmol min⁻¹) infused [mean (SEM) infused arm vs. control: 26.8 (5.3) vs. 2.6 ml min⁻¹ 100 ml⁻¹; P < 0.0001]. Dose-dependent flare and itch were demonstrated. Acute tolerance was not observed, with an increased FBF persisting throughout the infusion period. H₂-receptor antagonism significantly reduced FBF (mean (95% CI) difference from placebo at 30 nmol min⁻¹ histamine: −11.9 ml min⁻¹ 100 ml⁻¹ (−4.0, −19.8), P < 0.0001) and flare (mean (95% CI) difference from placebo: −403.7 cm² (−231.4, 576.0), P < 0.0001). No reduction in FBF or flare was observed in response to the H₁-receptor antagonist. Itch was unaffected by the treatments. Histamine did not stimulate vascular release of tissue plasminogen activator or von Willebrand factor.

Conclusion

Histamine causes dose-dependent vasodilatation, flare and itch in the human forearm. H₂-receptors are important in this process. Our results support further exploration of combined H₁- and H₂-receptor antagonist therapy in acute allergic syndromes.     

Development and Validation of a Stability-Indicating Capillary Electrophoresis Method for the Determination of Zolpidem Tartrate in Tablet Dosage Form with Positive Confirmation using 2D- and 3D-DAD Fingerprints

The aim of the current study was to develop a simple, precise, and accurate capillary zone electrophoresis method for the determination of zolpidem tartrate in tablet dosage form. Separation was conducted in normal polarity mode at 25°C, 22 kV, using hydrodynamic injection for 10 s. Separation was achieved using a background electrolyte of 20 mM disodium hydrogen phosphate adjusted with phosphoric acid (85%), pH at 5.50, and detection at 254 nm. Using the above optimized conditions, complete determination took place in less than 3 min using amiloride HCl as the internal standard. The method was linear over the range of 3–1000 μg mL⁻¹ with a correlation coefficient of 0.9999. Forced degradation studies were conducted by introducing a sample of zolpidem tartrate standard and pharmaceutical sample solutions to different forced degradation conditions, being neutral (water), basic (0.1 M NaOH), acidic (0.1 M HCl), oxidative (10% H₂O₂), temperature (60°C in oven for 3 days), and photolytic (exposure to UV light at 254 nm for 2 h). Degradation products resulting from the stress studies did not interfere with the detection of zolpidem tartrate and the assay can be considered stability-indicating.     

Comparison of inhibitors of superoxide generation in vascular smooth muscle cells

Background and purpose:

We compared the dose-dependent reductions in cellular superoxide anion (O₂⁻) by catalytic agents: superoxide dismutase (SOD), polyethylene glycol (PEG)-SOD and the nitroxide 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (tempol) with uncharacterized antioxidants: 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinate iron (III)(Fe-TTPS), (-)-cis-3,3′,4′,5,7-pentahydroxyflavane (2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol (-epicatechin), 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) and N-acetyl-L-cysteine (NAC) with the spin trap nitroblue tetrazolium (NBT) and with the vitamins or their analogues: ascorbate, α-tocopherol and 6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxy acid (trolox).

Experimental approach:

O₂⁻ was generated in primary cultures of angiotensin II-stimulated preglomerular vascular smooth muscle cells from spontaneously hypertensive rats and detected by lucigenin-enhanced chemiluminescence.

Key results:

SOD, PEG-SOD, NAC and tempol produced a similar maximum inhibition of O₂⁻ of 80–90%. -Epicatechin, NBT, ebselen and Fe-TTPS were significantly (P < 0.0125) less effective (50–70%), whereas trolox, α-tocopherol and ascorbate had little action even over 24 h of incubation (<31%). Effectiveness in disrupted and intact cells was similar for the permeable agents, PEG-SOD and tempol, but was enhanced for SOD. Generation of O₂⁻ was increased by NAC and NBT at low concentrations but reduced at high concentrations.

Conclusions and implications:

Maximum effectiveness against cellular production of O₂⁻ requires cell membrane permeability and catalytic action as exemplified by PEG-SOD or tempol. NAC and NBT have biphasic effects on O₂⁻ production. Vitamins C and E or analogues have low efficacy.     

Transmission FTIR derivative spectroscopy for estimation of furosemide in raw material and tablet dosage form

A Fourier transform infrared derivative spectroscopy (FTIR-DS) method has been developed for determining furosemide (FUR) in pharmaceutical solid dosage form. The method involves the extraction of FUR from tablets with N,N-dimethylformamide by sonication and direct measurement in liquid phase mode using a reduced path length cell. In general, the spectra were measured in transmission mode and the equipment was configured to collect a spectrum at 4 cm⁻¹ resolution and a 13 s collection time (10 scans co-added). The spectra were collected between 1400 cm⁻¹ and 450 cm⁻¹. Derivative spectroscopy was used for data processing and quantitative measurement using the peak area of the second order spectrum of the major spectral band found at 1165 cm⁻¹ (SO₂ stretching of FUR) with baseline correction. The method fulfilled most validation requirements in the 2 mg/mL and 20 mg/mL range, with a 0.9998 coefficient of determination obtained by simple calibration model, and a general coefficient of variation <2%. The mean recovery for the proposed assay method resulted within the (100±3)% over the 80%–120% range of the target concentration. The results agree with a pharmacopoeial method and, therefore, could be considered interchangeable.KEY WORDS: FTIR, Derivative spectroscopy, Furosemide, Frusemide, Pharmaceutical analysisAbbreviations: API, active pharmaceutical ingredient; DMF, N,N-dimethylformamide; DS, derivative spectroscopy; FTIR, Fourier transform infrared; FUR, furosemide or frusemide; HPLC, high performance liquid chromatographic; MIR, mid infrared     

Detection of exhaled hydrogen sulphide gas in rats exposed to intravenous sodium sulphide

Background and purpose:

Sodium sulphide (Na₂S) disassociates to sodium (Na⁺) hydrosulphide, anion (HS⁻) and hydrogen sulphide (H₂S) in aqueous solutions. Here we have established and characterized a method to detect H₂S gas in the exhaled breath of rats.

Experimental approach:

Male rats were anaesthetized with ketamine and xylazine, instrumented with intravenous (i.v.) jugular vein catheters, and a tube inserted into the trachea was connected to a pneumotach connected to a H₂S gas detector. Sodium sulphide, cysteine or the natural polysulphide compound diallyl disulphide were infused intravenously while the airway was monitored for exhaled H₂S real time.

Key results:

Exhaled sulphide concentration was calculated to be in the range of 0.4–11 ppm in response to i.v. infusion rates ranging between 0.3 and 1.1 mg·kg⁻¹·min⁻¹. When nitric oxide synthesis was inhibited with N^ω-nitro-L-arginine methyl ester the amount of H₂S exhaled during i.v. infusions of sodium sulphide was significantly increased compared with that obtained with the vehicle control. An increase in circulating nitric oxide using DETA NONOate [3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene] did not alter the levels of exhaled H₂S during an i.v. infusion of sodium sulphide. An i.v. bolus of L-cysteine, 1 g·kg⁻¹, and an i.v. infusion of the garlic derived natural compound diallyl disulphide, 1.8 mg·kg⁻¹·min⁻¹, also caused exhalation of H₂S gas.

Conclusions and implications:

This method has shown that significant amounts of H₂S are exhaled in rats during sodium sulphide infusions, and the amount exhaled can be modulated by various pharmacological interventions.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

The H2 Receptor Antagonist Nizatidine is a P-Glycoprotein Substrate: Characterization of its Intestinal Epithelial Cell Efflux Transport

The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H₂ receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05–10 mM in both apical–basolateral (AP–BL) and BL–AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL–AP than AP–BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC₅₀ of verapamil on nizatidine P-gp secretion was 1.2 × 10⁻² mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J_max = 5.7 × 10⁻³ nmol∙cm⁻²∙s⁻¹ and K_m = 2.2 mM) and one nonsaturable component (K_d = 7 × 10⁻⁴ μL∙cm⁻²∙s⁻¹). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V_max and K_m estimated for nizatidine P-gp-mediated secretion were 4 × 10⁻³ nmol∙cm⁻²∙s⁻¹ and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug–drug interactions.Key words: BCS class III drugs, caco-2 permeability, efflux transporters, intestinal absorption, nizatidine, P-glycoprotein     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

Synthesis,SAR Study and Evaluation of Mannich and Schiff Bases of Pyrazol-5(4H)-one Moiety Containing 3-(Hydrazinyl)-2-phenylquinazolin-4(3H)-one

In the present investigation, a series of 12 Mannich bases (QP1-12) and 5 Schiff bases (QSP1-5) of pyrazol-5(4H)-one moiety containing 3-(hydrazinyl)-2-phenylquinazolin-4(3H)-one has been synthesized and characterized by physicochemical as well as spectral means. The synthesized Mannich and Schiff bases were screened for their preliminary antimicrobial activity against Gram-positive and Gram-negative bacterial as well as fungal strains by the determination of zone of inhibition. Mannich bases (QP1-12) were found to be more potent antibacterial agents against Gram-positive bacteria, whereas Schiff bases (QSP1-5) were more potent against Gram-negative bacteria and fungi. Minimum inhibitory concentration result demonstrated that Mannich base compound (QP7) having ortho -OH and para -COOH group showed some improvement in antibacterial activity (minimum inhibitory concentration of 48.88×10⁻³ μM/ml) among the tested Gram-positive organisms and it also exhibit minimum inhibitory concentration of value of 12.22×10⁻³ μM/ml for Klebsiella pneumoniae. The antitubercular activity of synthesized compounds against Mycobacterium tuberculosis (H₃₇Rv) was determined using microplate alamar blue assay. Compound QP11 showed appreciable antitubercular activity (minimum inhibitory concentration of 6.49×10⁻³ μM/ml) which was more active than the standard drugs, ethambutol (minimum inhibitory concentration of 7.60×10⁻³ μM/ml) and ciprofloxacin (9.4×10⁻³ μM/ml). Compounds QP11, QP9, QSP1, QSP2, and QSP5 have good selective index and may be selected as a lead compound for the development of novel antitubercular agents.     

The analytical determination and electrochemiluminescence behavior of amoxicillin

A novel electrochemiluminescence (ECL) luminophor of amoxicillin was studied and found to generate ECL following the oxidation or reduction of amoxicillin. The amoxicillin oxidation state was also found to eliminate the reduction state, generating ECL. When solutions of amoxicillin were scanned between +1.5 V and −1.0 V with a graphite electrode in the presence of cetyltrimethyl ammonium bromide using KC1 as the supporting electrolyte, ECL emissions were observed at potentials of −0.7 V and +0.5 V. The ECL intensity at −0.7 V was enhanced by H₂O₂. Based on these findings, an ECL method for the determination of the amoxicillin concentration is proposed. The ECL intensities were linear with amoxicillin concentrations in the range of 1.8 × 10⁻⁸ g/mL to 2.5 × 10⁻⁷ g/mL, and the limit of detection (signal/noise = 3) was 5 × 10⁻⁹ g/mL. The florescence of amoxicillin had the greatest emission intensity in a neutral medium, with the emission wavelength dependent on the excitation wavelength. The experiments on the ECL mechanism for amoxicillin found that the electrochemical oxidation products of dissolved oxygen and active oxygen species contributed to the ECL process. The data also suggest that the hydroxyl group of amoxicillin contributed to its ECL emission.     

Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated,soluble AC-dependent cAMP amplification pathway common to GPCR signalling

BACKGROUND AND PURPOSE

H₂O₂ is widely understood to regulate intracellular signalling. In airway epithelia, H₂O₂ stimulates anion secretion primarily by activating an autocrine PGE₂ signalling pathway via EP₄ and EP₁ receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl⁻ secretion. This study investigated signalling downstream of the receptors activated by H₂O₂.

EXPERIMENTAL APPROACH

Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H₂O₂, an EP₄ receptor agonist or β₂-adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca²⁺]_I) changes were followed by microscopy using fura–2-loaded cells and PKA activation followed by FRET microscopy.

KEY RESULTS

Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H₂O₂ and EP₄ receptor-mediated CFTR activation in bronchial epithelia. H₂O₂ and EP₄ receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca²⁺]_i via Ca²⁺ store release (and not entry). Increased [Ca²⁺]_i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO₃⁻]. Ca²⁺-activated apical K_Ca1.1 channels and cAMP-activated basolateral K_V7.1 channels contributed to H₂O₂-stimulated anion currents. A similar Epac-mediated pathway was seen following β₂-adrenoceptor or forskolin stimulation.

CONCLUSIONS AND IMPLICATIONS

H₂O₂ initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca²⁺ crosstalk to activate sAC. The Epac-mediated Ca²⁺ signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.     

Efficient Adsorption of Deoxynivalenol by Porous Carbon Prepared from Soybean Dreg

A novel porous carbon adsorbent for the removal of deoxynivalenol was prepared from soybean dreg (SD). The new material was characterized by scanning electron microscopy equipped with energy dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM), Brunauer–Emmett–Teller (BET) analysis, N₂ adsorption/desorption measurement techniques, X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The specific surface area of the SDB-6-KOH was found to be 3655.95 m² g⁻¹, the pore volume was 1.936 cm³ g⁻¹ and the average pore size was 2.125 nm. The high specific surface area and effective functional groups of the carbon material promoted the adsorption of deoxynivalenol. By comparing the adsorption effect of SDB-6-X prepared with different activators (X: KOH, K₂CO₃, KHCO₃), SDB-6-KOH had the highest adsorption capacity. The maximum adsorption capacity of SDB-6-KOH to deoxynivalenol was 52.9877 µg mg⁻¹, and the removal efficiency reached 88.31% at 318 K. The adsorption kinetic and isotherm data were suitable for pseudo-second-order and Langmuir equations, and the results of this study show that the novel carbon material has excellent adsorptive ability and, thus, offers effective practical application potential for the removal of deoxynivalenol.     

Actions of hydrogen sulphide on ion transport across rat distal colon

Background and purpose:

The aim of this study was to identify the actions of H₂S on ion transport across rat distal colon.

Experimental approach:

Changes in short-circuit current (Isc) induced by the H₂S-donor, NaHS, were measured in Ussing chambers. Cytosolic Ca²⁺ concentration was evaluated using fura-2.

Key results:

NaHS concentration-dependently induced a change in Isc, that was only partially inhibited by the neurotoxin, tetrodotoxin. Lower concentrations (≤10⁻³ mol·L⁻¹) of NaHS induced a monophasic increase in Isc, whereas higher concentrations induced an additional, secondary fall of Isc, before a third phase when Isc rose again. Blockers of H₂S-producing enzymes (expression demonstrated immunohistochemically) decreased basal Isc, suggesting that endogenous production of H₂S contributes to spontaneous anion secretion. The positive Isc phases induced by NaHS were due to Cl⁻ secretion as shown by anion substitution and transport inhibitor experiments, whereas the transient negative Isc induced by higher concentrations of the H₂S-donor was inhibited by mucosal tetrapentylammonium suggesting a transient K⁺ secretion. When applied from the serosal side, glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and tetrapentylammonium, a blocker of Ca²⁺-dependent K⁺ channels, suppressed NaHS-induced Cl⁻ secretion suggesting different types of K⁺ channels are stimulated by the H₂S-donor. NaHS-induced increase in cytosolic Ca²⁺ concentration was confirmed in isolated, fura-2-loaded colonic crypts. This response was not dependent on extracellular Ca²⁺, but was inhibited by blockers of intracellular Ca²⁺ channels present on Ca²⁺ storage organelles.

Conclusions and implications:

H₂S induces colonic ion secretion by stimulation of apical as well as basolateral epithelial K⁺ channels.     

Tumor-targeted Gd-doped mesoporous Fe3O4 nanoparticles for T1/T2 MR imaging guided synergistic cancer therapy

In this study, a novel intelligent nanoplatform to integrate multiple imaging and therapeutic functions for targeted cancer theranostics. The nanoplatform, DOX@Gd-MFe₃O₄ NPs, was constructed Gd-doped mesoporous Fe₃O₄ nanoparticles following with the doxorubicin (DOX) loading in the mesopores of the NPs. The DOX@Gd-MFe₃O₄ NPs exhibited good properties in colloidal dispersity, photothermal conversion, NIR triggered drug release, and high T₁/T₂ relaxicity rate (r₁=9.64 mM⁻¹s⁻¹, r₂= 177.71 mM⁻¹s⁻¹). Benefiting from the high MR contrast, DOX@Gd-MFe₃O₄ NPs enabled simultaneous T₁/T₂ dual-modal MR imagining on 4T1 bearing mice in vivo and the MR contrast effect was further strengthened by external magnetic field. In addition, the DOX@Gd-MFe₃O₄ NPs revealed the strongest inhibition to the growth of 4T1 in vitro and in vivo under NIR irradiation and guidance of external magnetic field. Moreover, biosafety was also validated by in vitro and in vivo tests. Thus, the prepared DOX@Gd-MFe₃O₄ NPs would provide a promising intelligent nanoplatform for dual-modal MR imagining guided synergistic therapy in cancer theranostics.     

Decreased production of neuronal NOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

BACKGROUND AND PURPOSE

Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H₂O₂ is an important endothelium-derived relaxant factor in the mouse aorta. The role of H₂O₂ and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H₂O₂ contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE^−/−).

EXPERIMENTAL APPROACH

Changes in isometric tension were recorded on a myograph; simultaneously, NO and H₂O₂ were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms.

KEY RESULTS

Aortas from ApoE^−/− mice showed impaired vasodilatation paralleled by decreased NO and H₂O₂ production. Inhibition of nNOS with L-Arg^NO2-L-Dbu, knockdown of nNOS and catalase, which decomposes H₂O₂ into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H₂O₂ in wild-type animals, but had no effect in ApoE^−/− mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE^−/− mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE^−/− mice.

CONCLUSIONS AND IMPLICATIONS

Our data show that endothelial nNOS-derived H₂O₂ production is impaired and contributes to endothelial dysfunction in ApoE^−/− aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.