Dynamic of bacterial communities attached to lightened phytodetritus.

The effects of singlet oxygen ((1)O2) transfer to bacteria attached on phytodetritus were investigated under laboratory-controlled conditions. For this purpose, a nonaxenic culture of Emiliania huxleyi in late stationary phase was studied for bacterial viability. Our results indicated that only 9 ± 3% of attached bacteria were alive compared to 46 ± 23% for free bacteria in the E. huxleyi culture. Apparently, under conditions of low irradiance (36 W m(-2)), during the culture, the cumulative dose received (22,000 kJ m(-2)) was sufficiently important to induce an efficient (1)O2 transfer to attached bacteria during the senescence of E. huxleyi cells. At this stage, attached bacteria appeared to be dominated by pigmented bacteria (Maribacter, Roseobacter, Roseovarius), which should resist to (1)O2 stress probably due to their high contents of carotenoids. After subsequent irradiation of the culture until fully photodegradation of chlorophyll, DGGE analyses showed that the diversity of bacteria attached to E. huxleyi cells is modified by light. Photooxidative alterations of bacteria were confirmed by the increasing amounts of cis-vaccenic photoproducts (bacterial marker) per bacteria observed during irradiation time. Interestingly, preliminary chemotaxis experiments showed that Shewanella oneidensis considered here as a model of motile bacteria was attracted by phytodetritus producing or not (1)O2. This lack of repulsive effects could explain the high mortality rate of bacteria measured on E. huxleyi cells.

Blocking the QB-binding site of photosystem II by tenuazonic acid, a non-host-specific toxin of Alternaria alternata, activates singlet oxygen-mediated and EXECUTER-dependent signalling in Arabidopsis.

Necrotrophic fungal pathogens produce toxic compounds that induce cell death in infected plants. Often, the primary targets of these toxins and the way a plant responds to them are not known. In the present work, the effect of tenuazonic acid (TeA), a non-host-specific toxin of Alternaria alternata, on Arabidopsis thaliana has been analysed. TeA blocks the QB -binding site at the acceptor side of photosystem II (PSII). As a result, charge recombination at the reaction centre (RC) of PSII is expected to enhance the formation of the excited triplet state of the RC chlorophyll that promotes generation of singlet oxygen ((1)O2). (1)O2 activates a signalling pathway that depends on the two EXECUTER (EX) proteins EX1 and EX2 and triggers a programmed cell death response. In seedlings treated with TeA at half-inhibition concentration (1)O2-mediated and EX-dependent signalling is activated as indicated by the rapid and transient up-regulation of (1)O2-responsive genes in wild type, and its suppression in ex1/ex2 mutants. Lesion formation occurs when seedlings are exposed to higher concentrations of TeA for a longer period of time. Under these conditions, the programmed cell death response triggered by (1)O2-mediated and EX-dependent signalling is superimposed by other events that also contribute to lesion formation.

Singlet oxygen effects on lipid membranes: implications for the mechanism of action of broad-spectrum viral fusion inhibitors.

It was reported recently that a new aryl methyldiene rhodanine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.

Photoactivated hypericin induces downregulation of HER2 gene expression.

Photodynamic therapy is an alternative method for cancer treatment in which a photosensitizer exposed to a light source of suitable wavelength is excited and can subsequently react through free radical mechanisms. Recently, oxygen free radical-mediated changes in gene expression have been established. The present study shows the effect of photoactivated hypericin on the expression of the human epidermal growth factor receptor 2 (HER2) oncogene at both the mRNA and the protein level in SKBR-3 and MCF-7 breast adenocarcinoma cell lines. The photodynamic therapy-induced decrease in mRNA expression was reversed by the singlet oxygen scavenger trolox, which supports a role for singlet oxygen. In addition, prevention of the generation of reactive oxygen species by pretreatment with trolox effectively blocked the antiproliferation activity of photoactivated hypericin. These results may have important implications at least for recurrent breast cancer with HER2 expression alone or in combination with conventional therapies.

Photophysics and photochemistry of photodynamic therapy: fundamental aspects.

Photodynamic therapy (PDT) is a treatment modality for cancer and various other diseases. The clinical protocol covers the illumination of target cells (or tissue), which have been loaded with a photoactive drug (photosensitizer). In this review we describe the photophysical and primary photochemical processes that occur during PDT. Interaction of light with tissue results in attenuation of the incident light energy due to reflectance, absorption, scattering, and refraction. Refraction and reflection are reduced by perpendicular light application, whereas absorption can be minimized by the choice of a photosensitizer that absorbs in the far red region of the electromagnetic spectrum. Interaction of light and the photosensitizer can result in degradation, modification or relocalization of the drug, which differently affect the effectiveness of PDT. Photodynamic therapy itself, however, employs the light-induced chemical reactions of the activated photosensitizer (triplet state), resulting in the production of various reactive oxygen species, amongst them singlet oxygen as the primary photochemical product. Based on these considerations, the properties of an ideal photosensitizer for PDT are discussed. According to the clinical experience with PDT, it is proposed that the innovative concept of PDT is most successfully implemented into the mainstream of anticancer therapies by following an application-, i.e. tumor-centered approach with a focus on the actual clinical requirements of the respective tumor type.

RpoH(II) activates oxidative-stress defense systems and is controlled by RpoE in the singlet oxygen-dependent response in Rhodobacter sphaeroides.

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen ((1)O(2)). Here we show that the alternative sigma factor RpoH(II) is required for the expression of important defense factors and that deletion of rpoH(II) leads to increased sensitivity against exposure to (1)O(2) and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoH(II) is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH(II)-type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoH(II)-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, (1)O(2) scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH(II) in R. sphaeroides.

No single way to understand singlet oxygen signalling in plants.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Inactivation of cysteine and serine proteases by singlet oxygen.

The reaction of singlet oxygen with individual proteins is less well understood than that with other biological molecules. The inhibition of caspase 3 by singlet oxygen appears to involve the modification of a catalytic cysteine residue, since the reactivity of the sulfhydryl with alkylating agents decreased after singlet oxygen treatment. In addition to three cysteine proteases, two serine proteases were also found to be inhibited by singlet oxygen with a similar dose dependency, while an aspartate protease and a metalloprotease were not affected. The carbonyl content of these enzymes was elevated as the result of treatment with singlet oxygen. The catalytic center in serine proteases and cysteine proteases, in which catalytic reactions are based on similar mechanisms involving nucleophilic catalysis assisted by histidine as a general acid/base, can be expected to be modified by singlet oxygen and undergo inactivation.

A genetic approach towards elucidating the biological activity of different reactive oxygen species in Arabidopsis thaliana.

Plants are often exposed to external conditions that adversely affect their growth, development or productivity. Such unfavourable environmental stress factors may result in rapid and transient increases of intracellular concentrations of reactive oxygen species (ROS) that are chemically distinct and impact plants either by being cytotoxic or by acting as a signal. Because different ROS are generated simultaneously in different cellular and extracellular compartments, it is almost impossible to link a particular ROS to a specific stress response and to determine its mode of action. The conditional flu mutant of Arabidopsis has been used to determine the biological role of singlet oxygen. Immediately after a dark/light shift of the flu mutant, singlet oxygen is generated within the plastids activating several stress responses that include growth inhibition of mature plants and seedling lethality. These stress responses do not result from physicochemical damage caused by singlet oxygen, but are attributable to the activation of a genetically determined stress response programme triggered by the Executer1 protein. Singlet oxygen-mediated stress responses at the transcriptional level necessitate a retrograde transduction of signals from the chloroplast to the nucleus that activate distinct sets of genes different from those that are induced by superoxide/hydrogen peroxide. Hence, the biological activities of these two types of ROS are distinct from each other. Whether they act independently or interact is not known yet and is the topic of our current research.

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe.

Singlet oxygen (1O2) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4'(5')-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for 1O2. In this work, the probe is successfully used to characterize H2O2-dependent generation of 1O2 from saliva in real time. However, the yield of 1O2 is found to be very low, for example, being about 0.13 nmol from 200 microL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another 1O2 probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides 1O2, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H2O2. Furthermore, the present study shows that the selectivity of BAET for 1O2 is much higher than that of CLA and thus BAET is highly suited for the detection of 1O2 in the presence of other reactive oxygen species in biological systems.

The size of the population of weakly coupled chlorophyll pigments involved in thylakoid photoinhibition determined by steady-state fluorescence spectroscopy.

On the basis of experiments with singlet quenchers and in agreement with previous data, it is suggested that a population of energetically weakly coupled chlorophylls may play a central role in photoinhibition in vivo and in vitro. In the present study, we have used steady state fluorescence techniques to gain direct evidence for these uncoupled chlorophylls. Due to the presence of their emission maxima, near 650 nm and more prominently in the 670--675 nm interval both chlorophylls b and a seem to be involved. A straightforward mathematical model is developed to describe the data which allows us to conclude that the uncoupled/weakly coupled population size is in the range of 1--3 molecules per photosystem.

Regulation of hemostasis by singlet-oxygen (1DeltaO2*).

Hemostasis is the system of generation and destruction of thrombi. It consists of coagulation and thrombolysis and has a plasmatic part and a cellular one, the latter being the thrombocytes and endothelial cells for coagulation and the polymorphonuclear granulocytes (PMN) for thrombolysis. Main products of PMN are oxidants of the hypochlorite/chloramine-type that can generate the nonradical excited oxidant singlet molecular oxygen ((1)DeltaO(2)(*)). Physiologically, (1)DeltaO(2)(*) reacts with methionine and cysteine residues and with carbenic structures in lipids, generating dioxetanes, which upon disruption emit photons in the blue spectrum of light (380-450 nm). It modifies some important hemostasis components in blood: (1)DeltaO(2)(*) inactivates the factors I (fibrinogen), V, VIII, vWF, X, plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin. (1)DeltaO(2)(*) oxidation of plasminogen and fibrin facilitates their specific cleavage by plasminogen activators and plasmin. Furthermore,(1)DeltaO(2)(*)downregulates thrombocyte-function and upregulates PMN-function. Chloramines seem to be the main physiologic generators of (1)DeltaO(2)(*): in concentrations of 0.1-2 mM in blood they strongly inhibit coagulation and enhance thrombolysis. The biogenesis and reaction pattern of (1)DeltaO(2)(*) is of importance to understand the PMN-physiology in hemostasis, giving rise to new therapy forms of thromboatherothrombosis in man.

Singlet oxygen signaling: from intimate to global.

Reactive oxidizing species (ROS), such as hydrogen peroxide, nitric oxide, superoxide anion, and singlet oxygen, can damage cells as well as initiate responses such as new gene expression. The cell response evoked is strongly dependent on several factors. The subcellular location for formation of an ROS may be especially important for a highly reactive ROS, because it diffuses only a very short distance before reacting with a cellular molecule. How does a short-lived, locally acting ROS trigger responses at distant subcellular sites? This issue is discussed in light of a study of gene expression initiated by singlet oxygen, a ROS that exists for less than 100 ns in cells. Singlet oxygen is generated after a photosensitizer absorbs light energy and transfers the energy to molecular oxygen. To assess the role of singlet oxygen in light-induced gene expression in Arabidopsis, a mutant was used that accumulates the photosensitizer protochlorphyllide in chloroplasts. Three mechanisms are discussed that may connect the site of singlet oxygen formation to the signal transduction components: (i) direct oxidation of a signaling component by singlet oxygen, (ii) formation of oxidation products near the sites of singlet oxygen formation that diffuse to and react with signaling components, and (iii) alteration of the redox balance of the cell to a more oxidized state such that a greater proportion of a signaling pathway component is oxidized.

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes.

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a water-soluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkali-labile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

Singlet oxygen quenching activity of human serum.

Singlet oxygen is regarded as contributing to the pathogenesis of various diseases including light-induced skin disorders and inflammatory response. In this study, the correlation between singlet oxygen quenching activity (SOQA) of human serum and blood biochemistry or life-style was evaluated. Healthy volunteers were recruited and carried out a measurement of SOQA by using electron paramagnetic resonance (EPR) and a questionnaire survey about a smoking. It was demonstrated that major quenchers of singlet oxygen in serum are proteins, and small molecular anti-oxidants relatively play a minor role. SOQA of whole sera showed no correlation with protein concentration, but positively correlated with SOQA of small molecular fraction. In vitro studies demonstrated that the decrease of sulfhydryl groups by NO or superoxide significantly attenuated SOQA of albumin. Together, these results may imply that the underlying oxidative condition in each individual influences both small molecular antioxidant states and the sulfhydryl content of serum proteins. SOQA of sera from women with a smoking history was significantly lower compared to non-smoking women, suggesting that the smoking habit impaired the defense mechanism against singlet oxygen.

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes.

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.

The physiology and pharmacology of singlet oxygen.

Reactive oxygen species (ROS) are generated by many different cells. Singlet oxygen (1O(2)) and a reaction product of it, excited carbonyls (C=O*), are important ROS. 1O(2) and C=O* are nonradicalic and emit light (one photon/molecule) when returning to ground state oxygen. Especially activated polymorphonuclear neutrophil granulocytes (PMN) produce large amounts of 1O(2). Via activation of the respiratory burst (NADPH oxidase and myeloperoxidase) they synthesize hypochlorite (NaOCl) and chloramines (in particular N-chlorotaurine). Chloramines are selective and stable chemical generators of 1O(2). In the human organism, 1O(2) is both a signal and a weapon with therapeutic potency against very different pathogens, such as microbes, virus, cancer cells and thrombi. Chloramines at blood concentrations between 1 and 2 mmol/L inactivate lipid enveloped virus and chloramines at blood concentrations below 0.5 mmol/L, i.e. at oxidant concentrations that do not affect thrombocytes or hemostasis factors, act antithrombotically by activation of the physiologic PMN mediated fibrinolysis; this thrombolysis is of selective nature, i.e. it does not impair the hemostasis system of the patient allowing the antithrombotic treatment in patients where the current risky thrombolytic treatment is contraindicated. The action of 1O(2) might be compared to the signaling and destroying gunfire of soldiers directed against bandits at night, resulting in an autorecruitment of the physiological inflammatory response. Chloramines (such as the mild and untoxic oxidant chloramine T (N-chloro-p-toluene-sulfonamide)) and their signaling and destroying reaction product 1O(2) might be promising new therapeutic agents against a multitude of up to now refractory diseases.

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen.

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.

Neurospora crassa catalases, singlet oxygen and cell differentiation.

The morphogenetic transitions of the N. crassa asexual life cycle are responses to a hyperoxidant state in which probably singlet oxygen is generated. Induction of catalase activity and catalase oxidation by singlet oxygen are consequences of this recurrent hyperoxidant state. Here the biochemical properties and regulation of two large monofunctional catalases are reviewed, and a new catalase-peroxidase gene and activity is described. Catalase-3 is associated to growing and Catalase-1 to non-growing cells. Under stressful conditions one of these catalases is synthesized, depending on whether growth can be continued or a resistant cell has to be made. The catalase-peroxidase Catalase-2 was possibly derived from a bacterial enzyme. In contrast to the other catalases, Catalase-2 had catalase and peroxidase activity. Catalase-2 was expressed under conditions in which vacuolization of hyphae is observed. All three enzymes have a chlorin in its active site instead of ferroprotoheme IX and are resistant to molar concentrations of hydrogen peroxide. These and all other catalases tested so far are oxidized by singlet oxygen, probably at the heme moiety. The catalase activity is virtually unaffected by oxidation, but the enzymes are probably degraded more rapidly than the unmodified ones.