Analysis of chemokines and reactive oxygen species formation by rat and human neutrophils induced by microcystin-LA, -YR and -LR

Microcystins (MC), a family of heptapeptide toxins produced by some genera of Cyanobacteria, have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The present study reports the effects of MC-LA, MC-YR and MC-LR (1 and 1000 nM) on human and rat neutrophils functions in vitro. Cell viability, DNA fragmentation, mitochondrial membrane depolarization and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MC increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2 alpha beta (CINC-2 alpha beta) and extracellular ROS levels in human and rat neutrophils. Apart from neutrophil presence during the inflammatory process of MC-induced injury, our results suggest that hepatic neutrophil accumulation is further increased by MC-induced neutrophil-derived chemokine. (c) 2008 Elsevier Ltd. All rights reserved.

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Sterilization by pure oxygen plasma and by oxygen-hydrogen peroxide plasma: An efficacy study

Plasma is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperatures. The use of different parameters for this method of sterilization and the corresponding results were analyzed in this study. A low-pressure inductive discharge was used to study the plasma sterilization processes. Oxygen and a mixture of oxygen and hydrogen peroxide were used as plasma source gases. The efficacy of the processes using different combinations of parameters such as plasma-generation method, type of gas, pressure, gas flow rate, temperature, power, and exposure time was evaluated. Two phases were developed for the processes, one using pure oxygen and the other a mixture of gases. Bacillus subtilis var. niger ATCC 9372 (Bacillus atrophaeus) spores inoculated on glass coverslips were used as biological indicators to evaluate the efficacy of the processes. All cycles were carried out in triplicate for different sublethal exposure times to calculate the D value by the enumeration method. The pour-plate technique was used to quantify the spores. D values of between 8 and 3 min were obtained. Best results were achieved at high power levels (350 and 40oW) using pure oxygen, showing that plasma sterilization is a promising alternative to other sterilization methods. (c) 2007 Elsevier B.V. All rights reserved.

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria

We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extramitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extramitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.

Silencing of mitochondrial alternative oxidase gene of Aspergillus fumigatus enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.

Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex - A system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO) (ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Photoinduced Nitric Oxide and Singlet Oxygen Release from ZnPC Liposome Vehicle Associated with the Nitrosyl Ruthenium Complex: Synergistic Effects in Photodynamic Therapy Application

Under continuous photolysis at 675 nm, liposomal zinc phthalocyanine associated with nitrosyl ruthenium complex [Ru(NH.NHq)(tpy)NO](3+) showed the detection and quantification of nitric oxide (NO) and singlet oxygen ((1)O(2)) release. Photophysical and photochemical results demonstrated that the interaction between the nitrosyl ruthenium complex and the photosensitizer can enable an electron transfer process from the photosensitizer to the nitrosyl ruthenium complex which leads to NO release. Synergistic action of both photosensitizers and the nitrosyl ruthenium complex results in the production of reactive oxygen species and reactive nitrogen species, which is a potent oxidizing agent to many biological tissues, in particular neoplastic cells.

Iontophoretic transport of zinc phthalocyanine tetrasulfonic acid as a tool to improve drug topical delivery

Phthalocyanines have been used as systemic photosensitizers because of their high affinity towards tumour tissue, and the high rates of reactive oxygen species produced when they are irradiated during photodynamic therapy. However, the topical administration of these compounds is limited by their large size, poor hydrosolubility and ionic character. This study aimed to investigate the iontophoretic delivery of charged zinc phthalocyanine tetrasulfonic acid (ZnPcS(4)) from a hydrophilic gel to different skin layers by means of in-vitro and in-vivo studies. Six hours of passive administration was insufficient for ZnPcS(4) to cross the stratum corneum (SC) and to reach the epidermis and dermis. No positive effect was reached when anodal iontophoresis was performed, showing that the drug-electrode attraction effect was higher than the electro-osmosis contribution at a pH of 5.5. Cathodal iontophoresis, however, was able to transport significant amounts of the drug to the viable epidermis. In addition, the absence of NaCl in the formulation significantly increased (by five-fold) the amount of ZnPcS(4) that crossed the SC and accumulated in the epidermis and dermis. It was possible to visualize the drug accumulation in the follicle openings and in the epidermis, even after SC removal. In-vivo experiments in rat skin showed that these results were maintained in an in-vivo model, even with only 15 min of iontophoresis. In addition, confocal analysis of the treated skin showed a homogeneous distribution of ZnPcS(4) in the viable epidermis after this short period of cathodal iontophoresis. Anti-Cancer Drugs 22:783-793 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here. we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage. (C) 2009 Elsevier Inc. All rights reserved.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Self-assembled naphthalenediimide derivative films for light-assisted electrochemical reduction of oxygen

This naphthalene diimide derivative, DC18, forms highly conjugated semiconducting stacked assemblies over electrodes after electrochemical conditioning. These molecular materials are very efficient towards electrochemical photoreduction of oxygen under visible light.

Age and Method-dependent Variability of Predicted Oxygen Consumption in Congenital Heart Disease

Background and Purpose. There has been a lot of debate about the use of predicted oxygen consumption to calculate pulmonary vascular resistance using the Fick principle. We therefore comparatively analyzed predicted oxygen consumption in infants and children in specific age groups, using different methods (formulas), as an attempt to better understand the usefulness and limitations of predictions. Methods and Results. Four models (LaFarge & Miettinen, Bergstra et al., Lindahl, and Lundell et al.) were used to predict oxygen consumption in 200 acyanotic patients with congenital cardiac defects aged 0-2.0, > 2.0-4.0, > 4.0-6.0, and > 6.0-8.75 years (median 2.04 years). Significant differences were observed between the age groups (P < .001) and between the methods (P < .001), not related to diagnoses. Differences between methods were more impressive in the first age group (P < .01). In patients aged 0-2.0 years, the lowest values of oxygen consumption (corresponding to the highest estimates of pulmonary vascular resistance) were obtained with the method of Lindahl; above this age, any method except that of Lundell et al. Conclusions. Although measuring oxygen consumption is always preferable, a rational use of predictions, using different methods, may be of help in situations where measurements are definitely not possible.

Impact of clinical varicocele and testis size on seminal reactive oxygen species levels in a fertile population: a prospective controlled study

Objective: To investigate: 1) the impact of clinical varicocele on reactive oxygen species (ROS) levels in neat and washed semen in a proven fertile population; and 2) the correlation between ROS levels, testicular volume, and varicocele grade in the same population of fertile men. Design: Prospective controlled clinical study. Setting: Andrology laboratory at tertiary-care hospital. Patient(s): One hundred fourteen healthy fertile men (81 normal fertile and 33 fertile with clinical varicocele) and 30 infertile patients (control subjects). Intervention(s): Standard semen analysis and measurement of sperm ROS production. Main Outcome Measure(s): Seminal parameters, seminal ROS levels, seminal leukocyte levels, clinical varicocele, and testis size. Result(s): Thirty-three of the 11.4 (29%) fertile men had clinical varicocele (grade 1, n = 14; grade 2, n = 11; and grade 3, n = 8), and the remaining 81 (71%) had a normal physical examination. Levels of ROS and semen quality did not differ significantly between the fertile men with or without varicocele. No significant differences in ROS levels in neat and washed semen were observed compared with fertile men with grades 2 and 3 varicocele and with fertile men with varicocele grade 1. The ROS levels in neat and washed semen were not significantly correlated with varicocele grade in fertile men. No significant correlations between ROS levels and testis volume were observed between the fertile groups. Conclusion(s): The presence of clinical varicocele in fertile men is not associated with higher seminal ROS levels or abnormal semen parameters. Levels of ROS are not correlated with varicocele grade or testis volume in the same population of fertile men.

Comparison of Inhaled Nitric Oxide Versus Oxygen on Hemodynamics in Patients With Mitral Stenosis and Severe Pulmonary Hypertension After Mitral Valve Surgery

Pulmonary hypertension represents an important cause of morbidity and mortality in patients with mitral stenosis who undergo cardiac surgery, especially in the postoperative period. The aim of this study was to test the hypothesis that inhaled nitric oxide (iNO) would improve the hemodynamic effects and short-term clinical outcomes of patients with mitral stenosis and severe pulmonary hypertension who undergo cardiac surgery in a randomized, controlled study. Twenty-nine patients (4 men, 25 women; mean age 46 2 years) were randomly allocated to receive iNO (n = 14) or oxygen (n = 15) for 48 hours immediately after surgery. Hemodynamic data, the use of vasoactive drugs, duration of stay, and short-term complications were assessed. No differences in baseline characteristics were observed between the groups. After 24 and 48 hours, patients receiving iNO had a significantly greater increase in cardiac index compared to patients receiving oxygen (p < 0.0001). Pulmonary vascular resistance was also more significantly reduced in patients receiving iNO versus oxygen (-117 dyne/s/cm(5), 95% confidence interval 34 to 200, vs 40 dyne/s/cm5, 95% confidence interval 34 to 100, p = 0.005) at 48 hours. Patients in the iNO group used fewer systemic vasoactive drugs.(mean 2.1 +/- 0.14 vs 2.6 +/- 0.16, p = 0.046) and had a shorter intensive care unit stay (median 2 days, interquartile range 0.25, vs median 3 days, interquartile range 7, p = 0.02). In conclusion, iNO immediately after surgery in patients with mitral stenosis and severe pulmonary hypertension improves hemodynamics and may have short-term clinical benefits. (C) 2011 Elsevier Inc. All rights reserved. (Am J Cardiol 2011;107:1040-1045)