Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

Unique pharmacological actions of atypical neuroleptic quetiapine: possible role in cell cycle/fate control.

Quetiapine is an atypical neuroleptic with a pharmacological profile distinct from classic neuroleptics that function primarily via blockade of dopamine D2 receptors. In the United States, quetiapine is currently approved for treating patients with schizophrenia, major depression and bipolar I disorder. Despite its widespread use, its cellular effects remain elusive. To address possible mechanisms, we chronically treated mice with quetiapine, haloperidol or vehicle and examined quetiapine-specific gene expression change in the frontal cortex. Through microarray analysis, we observed that several groups of genes were differentially expressed upon exposure to quetiapine compared with haloperidol or vehicle; among them, Cdkn1a, the gene encoding p21, exhibited the greatest fold change relative to haloperidol. The quetiapine-induced downregulation of p21/Cdkn1a was confirmed by real-time polymerase chain reaction and in situ hybridization. Consistent with single gene-level analyses, functional group analyses also indicated that gene sets associated with cell cycle/fate were differentially regulated in the quetiapine-treated group. In cortical cell cultures treated with quetiapine, p21/Cdkn1a was significantly downregulated in oligodendrocyte precursor cells and neurons, but not in astrocytes. We propose that cell cycle-associated intervention by quetiapine in the frontal cortex may underlie a unique efficacy of quetiapine compared with typical neuroleptics.

Effects of activation of polymorphonuclear leukocytes on airway goblet cell mucin release in a co-culture system.

OBJECTIVE AND DESIGN: Effects of activated PMN on airway goblet cell mucin release were investigated using a co-culture system in which both tracheal surface epithelial (TSE) cells and PMN from hamsters were present. MATERIALS AND METHODS: TSE cells were metabolically labeled using (3)H-glucosamine and chased in the presence of PMN under various experimental designs. PMN were obtained from the bronchoalveolar lavage fluid of hamsters following intratracheal instillation of E. coli endotoxin. The amount of (3)H-mucin was measured by Sepharose CL-4B gel-filtration column chromatography. RESULTS: (i). activation of 10(6) PMN by fMLP (0.1 microM) and cytochalasin B (0.1 microM) resulted in production of both the estrolytic ("elastolytic") activity and superoxide, (ii). activation of PMN in the co-culture stimulated mucin release from TSE cells followed by a significant degradation of the released mucins, both of which were blocked in a dose-dependent fashion by pretreatment with alpha1-protease inhibitor, and (iii). generation of varying concentrations of superoxide in the TSE cell culture did not affect mucin release from TSE cells. CONCLUSION: In the co-culture system, activation of PMN results in release and degradation of mucins, both of which are almost entirely accounted for by serine proteases but not other cellular products such as superoxide.

Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation.

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)* during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected alpha-hydroxyethyl radical (CH(3)*CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH(3)*CHOH by NOS I was found to be Ca(2+)/calmodulin dependent. Superoxide dismutase prevented CH(3)*CHOH formation in the absence of L-arginine. However, in the presence of L-arginine, the production of CH(3)*CHOH was independent of O(2)* but dependent upon the concentration of L-arginine. Formation of CH(3)*CHOH was inhibited by substituting D-arginine for L-arginine, or inclusion of the NOS inhibitors N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe(5+)=O](3+)) in the absence of oxygen and Ca(2+)/calmodulin, and EtOH resulted in the formation of CH(3)*CHOH. NOS I was found to produce the corresponding alpha-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of L-arginine was responsible for catalyses of these secondary reactions.

The single-dose pharmacokinetics of midazolam and its primary metabolite in pediatric patients after oral and intravenous administration.

The first-dose pharmacokinetics of midazolam and its primary alpha-hydroxymetabolite were studied after single-dose administration. Eligible study patients were enrolled into one of three study arms: Arm I (midazolam/metabolite pharmacokinetic evaluation after oral administration of a syrup formulation), Arm II (the absolute bioavailability of midazolam syrup), and Arm III (midazolam and metabolite pharmacokinetics after IV administration). Complete blood sampling for pharmacokinetic analysis was available in 87 subjects. Midazolam absorption after administration of the oral syrupformulation was rapid, with adolescents absorbing the drug at approximately half the rate observed in younger children (ages 2 to < 12 years). Furthermore, midazolam t 1/2 was prolonged and CL/F reducedin adolescents as compared with younger children. Although the midazolam Vd/F appeared larger in the youngest age group after oral administration, this observation was not apparent after IV dosing, suggesting subject differences in bioavailability rather than distribution. Like midazolam, the disposition characteristics for a-hydroxymidazolam were also highly variable, with the greatest formation of metabolite (reflected by the AUC ratio) observed in children ages 2 to < 12 years. The A UC ratios of alpha-hydroxymidazolam to midazolam after IV dosing were similar across all age groups and were smaller than corresponding values following oral administration. The absolute bioavailability of midazolam averaged 36% with a very broad range (9%-71%). No relationship between midazolam bioavailability and age was observed. Overall, the disposition characteristics of midazolam and its a-hydroxy metabolite were highly variable, appeared independent of age and dose administered, and were linear over the dose range studied (0.25 to 1 mg/kg). These data suggest that an initial oral dose of 0.2 to 0.3 mg/kg should be adequateforsuccessful sedation of most pediatric patients. The inherent variability in midazolam bioavailability and metabolism underscores the importance of titrating midazolam dose to desired effect.

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies.

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-D-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO-Fe(MGD)2 and NO-Fe(DTCS)2 in mice in real time.

Mechanism of superoxide generation by neuronal nitric-oxide synthase.

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O-2, has until recently remained undefined. Herein, we investigated this relationship, observing O-2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced O-2 production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2 at concentrations that inhibited NO. formation from L-arginine. Taken together these data demonstrate that NOS I generates O-2 and the formation of this free radical occurs at the heme domain.

Effect of trifluoromethyl ketone-based elastase inhibitors on neutrophil function in vitro.

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha1-proteinase inhibitor (alpha1Pi). Nevertheless, under pathological conditions, alpha1i is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/alpha1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.

Use of nitroxides as NMR contrast enhancing agents for joints.

NMR imaging is a well-established technology for obtaining cross-sectional anatomic pictures of organs and tissues. In addition, NMR can provide valuable information about the physiologic state of organs and tissues, especially, as a consequence of cellular injury. With this in mind, NMR in combination with gadolinium-based contrast enhancing agents has been used to assist in the detection of abnormalities to joints as well as to evaluate the status of damage resulting from an injury to this site. We describe the synthesis of a new nitroxide, which is bioresistant to the one-electron reduction mediated by superoxide in the presence of cysteine. This model mimics the reduction of nitroxides by extracellular secretion of superoxide by PMA-stimulated neutrophils. With this nitroxide, we found, in the range from 15 to 17.5 mumoles, enhancement of an NMR image in the knee joint of rabbits. Of interest is the finding that the contrast image remained for at least 90 minutes. These results demonstrate the utility of nitroxides as contrast enhancing agents for NMR imaging of joints.

The use of fluorophore-containing spin traps as potential probes to localize free radicals in cells with fluorescence imaging methods.

Central to the study of free radical processes is the ability to identify and localize their cellular site of formation. Under the best of experimental conditions, spin trapping/ESR spectroscopy can only characterize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this article, we report on the development of a fluorophore-containing nitrone, alpha-[4-[5-((2-carboxy)phenyl)-5-hydroxy-4-oxo-3-phenyl)-2-pyrrolin+ -1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4). This nitrone (4) reacts with alpha-hydroxyethyl radical with a second order rate constant of 1.7 x 10(5) M-1 s-1 to give a characteristic ESR spectrum. However, we were unable to decrease the fluorescence emission, due in part to the small concentration of nitroxide generated from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found that only 12% of the nitroxide needs to be reduced to give an almost 400% increase in the fluorescent emission of (7). Our findings suggest new approaches to the development of various fluorophore-containing nitrones that can both characterize specific free radicals and localize their site of intracellular formation.

Does peroxynitrite generate hydroxyl radical?

Nitric oxide reacts with superoxide at a diffusion controlled rate to form peroxynitrite. Some studies have indicated that peroxynitrite underwent homolytic cleavage to give the highly toxic hydroxyl radical, while others have suggested that the decomposition of peroxynitrite did not generate hydroxyl radical. Because of this controversy, the fate of peroxynitrite decomposition was investigated using electron spin resonance (EPR) spectroscopy in combination with spin trapping technique. Utilizing 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol as a spin trapping system, we found that peroxynitrite, at physiological pH in either the absence or the presence of chelated iron, will produce hydroxyl radical. Further quantification experiments indicated that the yield of hydroxyl radical formation was only about 1-4%. Since the concentration of hydroxyl radical produced is so low, the cytotoxicity mediated by peroxynitrite might not be due to the formation of this free radical.

Effect of anti-inflammatory drugs on myeloperoxidase-dependent hydroxyl radical generation by human neutrophils.

Neutrophils comprise a group of leukocytes that play a pivotal role in inflammation and vascular diseases like ischemia/reperfusion. These activated phagocytic cells are drawn to the site of injury, secreting superoxide and other oxidants derived from the formation of this free radical. This series of events frequently results in localized tissue damage. Surprisingly, free radical scavengers frequently offer only minimal relief. Why this is so may be due, in part, to our limited understanding of mechanisms that govern generation of free radicals in these settings. Although the metal ion-catalyzed Haber-Weiss reaction is considered the classical pathway for neutrophil-derived hydroxyl radical, an alternative mechanism, such as the myeloperoxidase-dependent pathway, may undoubtedly contribute to the formation of this free radical by stimulated neutrophils. In this study, we explored this possibility by investigating the role of different classes of anti-inflammatory drugs to ameliorate hydroxyl radical generation via the myeloperoxidase-dependent pathway. In this paper, we report that meclofenamic acid inhibited myeloperoxidase-dependent hydroxyl radical generation through scavenging of hypochlorous acid and not by direct inhibition of myeloperoxidase. The importance of these results with regard to the clinical efficacy of this anti-inflammatory compound remains to be determined as studies into the significance of myeloperoxidase-dependent hydroxyl radical formation in inflammatory tissue injury continue.

Free radicals and phagocytic cells.

Phagocytes mediate their innate immunological response by releasing products that damage invading microorganisms. These products include proteins such as lysozyme, peroxidases, and elastase as well as reactive oxygen species such as superoxide, hydrogen peroxide, hypohalous acid, and hydroxyl radical. Although it is clear that many phagocytic secretory products have direct cytotoxic potential, understanding is limited of how multiple products interact to generate and modulate the cytotoxic response. This review focuses on recent findings that elucidate the biochemical nature of secretory product interaction in the formation of free radicals, particularly the highly reactive hydroxyl radical. The possible role of these reactions in phagocyte microbicidal activity and inflammatory tissue injury is discussed.

Biological studies of a nitroso compound that releases nitric oxide upon illumination.

2-Methyl-2-nitrosopropane (MNP) has long been known to undergo photochemical and thermal decomposition, generating di-tert-butyl nitroxide, in organic solvent. The present study was undertaken to demonstrate that MNP can be used as a caged-nitric oxide (NO), which can liberate NO upon illumination. Photolysis of MNP leads to the generation of tert-butyl radical and NO, as detected by spin-trapping/ESR spectroscopy and by oxyhemoglobin/visible spectroscopy, respectively. Using soluble guanylate cyclase in neuroblastoma N1E-115 cells as an NO target, we found that MNP in the presence of light caused a dose- and time-dependent increase in cGMP. Finally, illumination of a solution of MNP was also found to induce relaxation of preconstricted isolated rat pulmonary artery rings. These studies demonstrated that MNP can be useful biochemical research tool for delivering NO in a controlled manner, by using light.

Can nitric oxide be spin trapped by nitrone and nitroso compounds?

Increasing interest in the study of nitric oxide (NO.) in many facets of biological research necessitates a search for accurate techniques to directly identify the free radical. One recently employed strategy for NO. detection is the method of electron spin resonance (ESR) used in combination with nitrone and nitroso spin traps. Applying this technique to our studies with nitric oxide synthase (NOS), we found that NO. generated directly from the enzyme system could not be detected. Further investigation revealed that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) inhibited NO. generation by NOS at concentrations used for spin trapping. Reexamining the ability of various nitrones and DBNBS to spin trap authentic NO. dissolved in buffer, we obtained ESR spectra similar to those previously reported for the spin trap DBNBS. However, continuing our studies with 15NO. and N-hydroxylamine, we found these spectra to be artifactual. Our results emphasize the need to synthesize new spin traps, since currently available compounds are not capable of spin trapping NO. generated by NOS.

A kinetic approach to the selection of a sensitive spin trapping system for the detection of hydroxyl radical.

The spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) alone, as well as DMPO or N-tert-butyl-alpha-phenylnitrone (PBN) in the presence of excess dimethyl sulfoxide (Me2SO), have been used as spin trapping systems for the detection of hydroxyl radical. However, the instability of DMPO and many of its corresponding spin-trapped adducts has limited the usefulness of this spin trap, particularly in biological systems. Spin trapping of multiple free radicals by the PBN/Me2SO system may undermine the sensitivity of this method to detect small, yet biologically significant amounts of hydroxyl radical. The present study was undertaken to select a spin trapping system with greater sensitivity and selectivity toward.OH than DMPO, DMPO/Me2SO, or PBN/Me2SO. We report that alpha-hydroxyethyl radical, resulting from the reaction of photolytically generated.OH with excess ethanol is spin trapped by 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) with a second-order rate constant nearly 10-fold greater than that for DMPO or PBN. In contrast to DMPO spin-trapped adducts, the alpha-hydroxyethyl radical adduct of 4-POBN, 4-POBN-CH(CH3)OH, is resistant to reduction by superoxide, even in the presence of cysteine. The efficiency of spin trapping and the marked stability of the resulting spin-trapped adduct confer a high degree of sensitivity and demonstrate the potential application of 4-POBN/ETOH toward the detection of hydroxyl radical in biological systems.