Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores of Phytophthora parasitica var nicotianae

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O₂⁻) production based on reduction of the tetrazolium dye sodium,3′-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO₂^·/O₂⁻) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO₂^·/O₂⁻ production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO₂^·/O₂⁻ was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10⁻¹⁵ mol min⁻¹ cell⁻¹ were observed. The HO₂^·/O₂⁻ scavengers O₂⁻ dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O₂⁻ production is a necessary precursor to the HR.     

Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants

Chilling temperatures (5°C) and high irradiance (1000 microeinsteins per square meter per second) were used to induce photooxidation in detached leaves of cucumber (Cucumis sativus L.), a chilling-sensitive plant. Chlorophyll a, chlorophyll b, β carotene, and three xanthophylls were degraded in a light-dependent fashion at essentially the same rate. Lipid peroxidation (measured as ethane evolution) showed an O₂ dependency. The levels of three endogenous antioxidants, ascorbate, reduced glutathione, and α tocopherol, all showed an irradiance-dependent decline. α-Tocopherol was the first antioxidant affected and appeared to be the only antioxidant that could be implicated in long-term protection of the photosynthetic pigments. Results from the application of antioxidants having relative selectivity for ¹O₂, O₂⁻, or OH indicated that both ¹O₂ and O₂⁻ were involved in the chilling- and light-induced lipid peroxidation which accompanied photooxidation. Application of D₂O (which enhances the lifetime of ¹O₂) corroborated these results. Chilling under high light produced no evidence of photooxidative damage in detached leaves of chilling-resistant pea (Pisum sativum L.). Our results suggest a fundamental difference in the ability of pea to reduce the destructive effects of free-radical and ¹O₂ production in chloroplasts during chilling in high light.     

Chilling-Enhanced Photooxidation : The Peroxidative Destruction of Lipids during Chilling Injury to Photosynthesis and Ultrastructure

Chilling-induced photooxidation was studied in detached leaves of chilling-sensitive (CS) cucumber (Cucumis sativus L.) and chilling resistant (CR) pea (Pisum sativum L.). The rates of photosynthesis and respiration, measured as O₂ exchange, were found to be comparable in the two species over a temperature range of 5 to 35°C. Chilling at 5°C for 12 hours in high light (1000 microeinsteins per square meter per second) decreased CO₂ uptake 75% in detached pea leaves whereas CO₂ uptake by cucumber was reduced to zero within 2 hours. Respiration was unaffected in either species by the chilling and light treatment. Although ultrastructural alterations were apparent in chloroplasts of both species, cucumber's were affected sooner and more severely. The mechanism of photooxidative lipid peroxidation was investigated by following the production of ethane gas under a variety of conditions. Maximum ethane production occurred in the CS cucumber at low temperature (5°C) and high light (1000 microeinsteins per square meter per second). Atrazine, an inhibitor of photosynthetic electron transport, almost completely halted this chilling- and light-induced ethane production. These data, taken with those reported in an accompanying article (RR Wise, AW Naylor 1986 Plant Physiol 83: 278-282) suggest that the superoxide anion radical is generated in cucumber chloroplasts (probably via a Mehler-type reaction) during chilling-enhanced photooxidation. Parallel experiments were conducted on pea, a CR species. Detached pea leaves could only be made to generate ethane in the cold and light if they were pretreated with the herbicide parquat, a known effector of O₂⁻ production. Even so, pea showed no lipid peroxidation for 6 hours, at which time ethane production began and was at a rate equal to that for the chilled and irradiated cucumber leaves. The results indicate that pea has an endogenous mechanism(s) for the removal of toxic oxygen species prior to lipid peroxidation. This mechanism breaks down in pea after 6 hours in the cold, light, and the presence of paraquat.     

TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO₂) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO₂ photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O₂·⁻), inhibited this effect by half, showing us that O₂·⁻ radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO₂, respectively, which is accounted for by the cytotoxic effect of TiO₂. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO₂ increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O₂·⁻ on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.     

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal,a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH₄ ⁺ ion, and H₂O₂ coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O₂ ^•− and HO^• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E₀ values = −0.51 and −1.0 V) to ferricytochrome c (E₀ = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH₄ ⁺ ion. In the presence of oxygen, aminoacetone enoyl and O₂ ^•− radicals propagate aminoacetone oxidation to methylglyoxal and H₂O₂. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.     

Fusarium Wilt Suppression and Agglutinability of Pseudomonas putida

Mutants of Pseudomonas putida (Agg⁻) that lack the ability to agglutinate with components present in washes of bean and cucumber roots showed limited potential to protect cucumber plants against Fusarium oxysporum f. sp. cucumerinum. However, a higher level of protection was observed against Fusarium wilt in cucumber plants coinoculated with the parental bacterium (Agg⁺), which was agglutinable. The Agg⁻ mutants did not colonize the roots of cucumber plants as extensively as the Agg⁺ parental isolate did. In competition experiments involving bean roots inoculated with a mixture of Agg⁺ and Agg⁻ bacteria, the Agg⁺ strains colonized roots to a greater extent than the Agg⁻ cells did. These data suggest that the Agg⁺ phenotype provides additional interactions that aid in the beneficial character of P. putida.     

Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H₂O₂ only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O₂⁻, H₂O₂, OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.     

Delay of Senescence of Detached Cucumber Cotyledons by Triadimefon

Changes in contents of reactive oxygen species (O₂ ⁻ and H₂O₂) and non-enzymatic antioxidants, activities of antioxidant enzymes and lipid peroxidation were investigated during senescence of detached cucumber cotyledons dipped in water (control) and 20 mg dm⁻³ triadimefon (TDM). O₂ ⁻ and H₂O₂ accumulation and lipid peroxidation were observed during senescence of cucumber cotyledons, which coincided with a drop in the contents of carotenoids (Car) and ascorbic acid (AsA), and the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and an increase in the activity of peroxidase (POD). However, TDM could significantly inhibit the accumulation of O₂ ⁻ and H₂O₂, and lipid peroxidation by preventing the decrease of CAT, APX, Car and AsA and the increase of POD, while TDM had little effect on SOD activity during the senescence. Therefore we can draw a conclusion that TDM protects the membrane system and retards the senescence of detached cucumber cotyledons. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe²⁺ and H₂O₂ in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe³⁺ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe³⁺ at a rate constant of 4.5 × 10³ M⁻¹s⁻¹ at pH 4.0. Fe²⁺ is also very stable at a low pH. H₂O₂ generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe³⁺. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe³⁺. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Contribution of Oxidative Stress to the Development of Cold-Induced Damage to Leaves of Chilling-Sensitive Plants: 1. Reactive Oxygen Species Formation during Plant Chilling

Changes in the levels of superoxide anion radical and total peroxides were studied immediately after the chilling of 7–11-day-old seedlings of maize (Zea mays L.), cucumber (Cucumis sativus L.), millet (Panicum miliaceum L.), and etiolated potato (Solanum tuberosum L.) shoots at 2°C for 1–24 h and one day after 24-h chilling. A short-term (1 h) chilling of chilling-sensitive plants resulted in the 2.4–7.5-fold acceleration of the O^– ₂ generation. A longer chilling period reduced somewhat the rate of O^– ₂ generation, but this rate did not achieve the control level. The highest level of H₂O₂ was observed after 2-h chilling with its subsequent lowering. In the cold-tolerant potato, the levels of O^– ₂ and peroxides reduced after chilling. The rate of lipid peroxidation (an index characterizing cold-induced membrane damage) increased gradually with the lengthening of the chilling period. Reactive oxygen species are supposed to be involved in the induction of the oxidative stress during chilling of chilling-sensitive plants and in the triggering of cold-induced damage.     

The effects of dopamine on antioxidant enzymes activities and reactive oxygen species levels in soybean roots

In the current work, we investigated the effects of dopamine, an neurotransmitter found in several plant species on antioxidant enzyme activities and ROS in soybean (Glycine max L. Merrill) roots. The effects of dopamine on SOD, CAT and POD activities, as well as H₂O₂, O₂^•−, melanin contents and lipid peroxidation were evaluated. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.1 to 1.0 mM dopamine, in a growth chamber (25°C, 12 h photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 h. Significant increases in melanin content were observed. The levels of ROS and lipid peroxidation decreased at all concentrations of dopamine tested. The SOD activity increased significantly under the action of dopamine, while CT activity was inhibited and POD activity was unaffected. The results suggest a close relationship between a possible antioxidant activity of dopamine and melanin and activation of SOD, reducing the levels of ROS and damage on membranes of soybean roots.     

Bactericidal Activity of Photocatalytic TiO2 Reaction: toward an Understanding of Its Killing Mechanism

When titanium dioxide (TiO₂) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO₂ photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells⁻¹ after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO₂. We concluded that TiO₂ photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.     

Seasonal Mesophotic Coral Bleaching of Stylophora pistillata in the Northern Red Sea

Coral bleaching occurs when environmental stress induces breakdown of the coral-algae symbiosis and the host initiates algae expulsion. Two types of coral bleaching had been thoroughly discussed in the scientific literature; the first is primarily associated with mass coral bleaching events; the second is a seasonal loss of algae and/or pigments. Here, we describe a phenomenon that has been witnessed for repeated summers in the mesophotic zone (40–63 m) in the northern Red Sea: seasonal bleaching and recovery of several hermatypic coral species. In this study, we followed the recurring bleaching process of the common coral Stylophora pistillata. Bleaching occurred from April to September with a 66% decline in chlorophyll a concentration, while recovery began in October. Using aquarium and transplantation experiments, we explored environmental factors such as temperature, photon flux density and heterotrophic food availability. Our experiments and observations did not yield one single factor, alone, responsible for the seasonal bleaching. The dinoflagellate symbionts (of the genus Symbiodinium) in shallow (5 m) Stylophora pistillata were found to have a net photosynthetic rate of 56.98–92.19 µmol O₂ cm⁻² day⁻¹. However, those from mesophotic depth (60 m) during months when they are not bleached are net consumers of oxygen having a net photosynthetic rate between −12.86 - (−10.24) µmol O₂ cm⁻² day⁻¹. But during months when these mesophotic corals are partially-bleached, they yielded higher net production, between −2.83–0.76 µmol O₂ cm⁻² day⁻¹. This study opens research questions as to why mesophotic zooxanthellae are more successfully meeting the corals metabolic requirements when Chl a concentration decreases by over 60% during summer and early fall.     

ESR evidence of the formation of a new superoxide complex of tetra-p-tolyporphyrinatocobalt(II) in aprotic solvents

The reactions of the superoxide ion (O₂⁻) with tetra-p-tolyporphyrinatocobalt(II) [Co(II)TTP] in dimethyl sulfoxide(DMSO) have been investigated by use of electron spin resonance (ESR) spectroscopy. In the absence of oxygen, Co(II)TTP in DMSO gives the DMSO adduct, Co(II)(TPP)(DMSO). When this DMSO adduct is exposed to air, an oxygen complex, Co(II)(TTP)(DMSO)(O₂), is formed in which the binding state between Co(II) and O₂ has been considered formally as Co(III)O₂⁻. When the superoxide ion (O₂⁻ is added to this oxygen complex, a new superoxide complex, Co(II)(TTP)(O₂⁻)₂, is formed. The same superoxide adduct is formed by the reaction of O₂⁻ with Co(II)TTP in the absence of oxygen.     

Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2)

Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO₂ per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F_O) was increased and induced fluorescence ([F_P-F_I]/F_O) was reduced. The effect was greatest in the undroughted plants grown at 660 μl CO₂ L⁻¹. By week 22 at 330 μl CO₂ L⁻¹ acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 μl CO₂ L⁻¹. The light saturated rate of photosynthesis of needles with adequate P was higher at 660 μl CO₂ L⁻¹ than at 330 μl CO₂ L⁻¹, whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO₂ concentration. The average growth increase due to CO₂ enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 μl CO₂ L⁻¹, there was a reduction in the maximal rate of quenching of fluorescence (R_Q) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 μl CO₂ L⁻¹ this response was eliminated showing that CO₂ enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO₂ enrichment was 37% in drought stressed plants and 19% in unstressed plants.     

Oxygen Microelectrode That Is Insensitive to Medium Chemical Composition: Use in an Acid Microbial Mat Dominated by Cyanidium caldarium

A novel oxygen microelectrode with a tip diameter of 2 to 20 μm was constructed which could function satisfactorily under a variety of environmental conditions and in a variety of media, including human blood serum, citric acid at pH 2.5, moist air, and paraffin oil. Measurement of oxygen by this electrode does not require stirring of the medium. Electrodes could be made so that the 90% response time necessary to detect changes in oxygen concentration was less than 0.2 s, and response was linear with oxygen concentration. To demonstrate the performance of the electrode, oxygen and photosynthesis profiles of an acid microbial mat (pH 2.8) dominated by the eucaryotic alga Cyanidium caldarium were made. Photosynthetic rates as high as 95 mmol of O₂ dm⁻³ h⁻¹ were measured within the most active 0.1-mm layer, which was ca. 0.2 mm below the surface of the microbial mat. The total photosynthetic activity was 47 mmol of O₂ m⁻² h⁻¹. Vertical profiles of photosynthesis at different light intensities showed that the microalgae within the mat were not photoinhibited at bright sunlight (2,090 μEinsteins m⁻² s⁻¹).     

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M₂ muscarinic receptor (M₂R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K⁺ channel I_KACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4^−/−, Rgs6^−/−, and Rgs4^−/−:Rgs6^−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M₂R-I_KACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M₂R-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6^−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6^−/− and Gβ5^−/− mice, suggest that the partial rescue of phenotypes in Rgs4^−/−:Rgs6^−/− mice is attributable to another R7 RGS protein whose influence on M₂R-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O₂ ^•−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O₂ ^•− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O₂ ^•− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O₂ ^•− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O₂ ^•− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O₂ ^•− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.     

Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H₂O₂) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H₂O₂ levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW⁻¹, 140% greater than that of the control. Activities of POD (421.3 U gFW⁻¹), PPO (508.8 U gFW⁻¹) and PAL (707.3 U gFW⁻¹) were also greater in the treated plants than in the controls (103.0 U gFW⁻¹, 166.0 U gFW⁻¹ and 309.4 U gFW⁻¹, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H₂O₂ concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.