Adenosine A₂a receptors and O₂ sensing in development

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O? sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(?A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(?A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(?A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(?A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(?A) receptors play virtually no role in O? sensing by the carotid bodies, but brain A(?A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(?A) receptors have been implicated in O? sensing by carotid glomus cells, while central A(?A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(?A) receptors are crucially involved in the transduction mechanisms of O? sensing in fetal carotid bodies and brains. Postnatally, central A(?A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O? sensing in carotid chemoreceptors, particularly in developing lambs.     

Effect of exercise training on nitric oxide and superoxide/H₂O₂ signaling pathways in collateral-dependent porcine coronary arterioles

Endothelial nitric oxide (NO) synthase (NOS) has been shown to contribute to enhanced vascular function after exercise training. Recent studies have revealed that relatively low concentrations of reactive oxygen species can contribute to endothelium-dependent vasodilation under physiological conditions. We tested the hypothesis that exercise training enhances endothelial function via endothelium-derived vasodilators, NO and superoxide/H(2)O(2), in the underlying setting of chronic coronary artery occlusion. An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. At 8 wk postoperatively, pigs were randomly assigned to sedentary (pen-confined) or exercise-training (treadmill-run: 5 days/wk for 14 wk) regimens. Exercise training significantly enhanced concentration-dependent, bradykinin-mediated dilation in cannulated collateral-dependent arterioles (～130 μm diameter) compared with sedentary pigs. NOS inhibition reversed training-enhanced dilation at low bradykinin concentrations in collateral-dependent arterioles, although increased dilation persisted at higher bradykinin concentrations. Total and phosphorylated (Ser(1179)) endothelial NOS protein levels were significantly increased in arterioles from collateral-dependent compared with the nonoccluded region, independent of exercise. The H(2)O(2) scavenger polyethylene glycol-catalase abolished the training-enhanced bradykinin-mediated dilation in collateral-dependent arterioles; similar results were observed with the SOD inhibitor diethyldithiocarbamate. Fluorescence measures of bradykinin-stimulated H(2)O(2) levels were significantly increased by exercise training, independent of occlusion. The NADPH inhibitor apocynin significantly attenuated bradykinin-mediated dilation in arterioles of exercise-trained, but not sedentary, pigs and was associated with significantly increased protein levels of the NADPH subunit p67phox. These data provide evidence that, in addition to NO, the superoxide/H(2)O(2) signaling pathway significantly contributes to exercise training-enhanced endothelium-mediated dilation in collateral-dependent coronary arterioles.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Synthesis of functional SiO₂-coated graphene oxide nanosheets decorated with Ag nanoparticles for H₂O₂ and glucose detection

In this paper, we report on the first preparation of well-defined SiO(2)-coated graphene oxide (GO) nanosheets (SiO(2)/GO) without prior GO functionalization by combining sonication with sol-gel technique. The functional SiO(2)/GO nanocomposites (F-SiO(2)/GO) obtained by surface functionalization with NH(2) group were subsequently employed as a support for loading Ag nanoparticles (AgNPs) to synthesize AgNP-decorated F-SiO(2)/GO nanosheets (AgNP/F-SiO(2)/GO) by two different routes: (1) direct adsorption of preformed, negatively charged AgNPs; (2) in situ chemical reduction of silver salts. The morphologies of these nanocomposites were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is found that the resultant AgNP/F-SiO(2)/GO exhibits remarkable catalytic performance for H(2)O(2) reduction. This H(2)O(2) sensor has a fast amperometric response time of less than 2s. The linear range is estimated to be from 1×10(-4) M to 0.26 M (r=0.998) and the detection limit is estimated to be 4 × 10(-6) M at a signal-to-noise ratio of 3, respectively. We also fabricated a glucose biosensor by immobilizing glucose oxidase (GOD) into AgNP/F-SiO(2)/GO nanocomposite-modified glassy carbon electrode (GCE) for glucose detection. Our study demonstrates that the resultant glucose biosensor can be used for the glucose detection in human blood serum.     

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.     

Catalytic conversion of xylose to furfural over the solid acid SO₄ ²⁻-/ZrO₂-Al₂O₃/SBA-15 catalysts

Al-promoted SO? 2?-/ZrO?SBA-15 catalysts were prepared and characterized by XRD, BET, ICP and NH?-TPD techniques. The influence of introducing aluminum on the structure and surface properties of the catalyst and the catalytic activity for dehydration of xylose to furfural has been investigated. The introduction of the Al stabilizes the tetragonal phase of the ZrO? and thus increases the number and intensity of acid sites. Based on the characterization of the deactivated catalyst, the accumulation of byproducts is the main reason for the deactivation of the catalyst. Regeneration with H?O? can completely recover the catalytic activity of the deactivated catalyst.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

CO₂ utilizing microbes--a comprehensive review

CO₂ fixing microbes are the species primarily engaged in complexing the inorganic carbon dioxide to organic carbon compounds. There are many microorganisms from archaeal and bacterial domain that can fix carbon dioxide through six known CO₂ fixing pathways. These organisms are ubiquitous and can survive in wide range of aerobic and anaerobic habitats. This review focuses on the prior research, that has been conducted in this field and presents a summarized overview of all the mechanisms (along with their genes and enzymes) used by these microbes for CO₂ incorporation. In addition, this review provides a better understanding of diversity and taxonomy of CO₂ fixing microorganisms. The information presented here will motivate researchers to further explore the diversity of CO₂ fixing microorganisms as well as to decipher the underlying mechanisms of CO₂ utilization.     

VO₂ requirements of boxing exercises

The purpose of this study was to quantify the physiological requirements of various boxing exercises such as sparring, pad work, and punching bag. Because it was not possible to measure the oxygen uptake (VO?) of "true" sparring with a collecting gas valve in the face, we developed and validated a method to measure VO? of "true" sparring based on "postexercise" measurements. Nine experienced male amateur boxers (Mean ± SD: age = 22.0 ± 3.5 years, height = 176.0 ± 8.0 cm, weight = 71.4 ± 10.9 kg, number of fights = 13.0 ± 9.5) of regional and provincial level volunteered to participate in 3 testing sessions: (a) maximal treadmill test in the LAB, (b) standardized boxing training in the GYM, and (c) standardized boxing exercises in the LAB. Measures of VO?, heart rate (HR), blood lactate concentration [LA], rated perceived exertion level, and punching frequencies were collected. VO? values of 43.4 ± 5.9, 41.1 ± 5.1, 24.7 ± 6.1, 30.4 ± 5.8, and 38.3 ± 6.5 ml·kg?1·min?1 were obtained, which represent 69.7 ± 8.0, 66.1 ± 8.0, 39.8 ± 10.4, 48.8 ± 8.5, and 61.7 ± 10.3%VO?peak for sparring, pad work, and punching bag at 60, 120, and 180 b·min?1, respectively. Except for lower VO? values for punching the bag at 60 and 120 b·min?1 (p < 0.05), there was no VO? difference between exercises. Similar pattern was obtained for %HRmax with respective values of 85.5 ± 5.9, 83.6 ± 6.3, 67.5 ± 3.5, 74.8 ± 5.9, and 83.0 ± 6.0. Finally, sparring %HRmax and [LA] were slightly higher in the GYM (91.7 ± 4.3 and 9.4 ± 2.2 mmol·L?1) vs. LAB (85.5 ± 5.9 and 6.1 ± 2.3 mmol·L?1). Thus, in this study simulated LAB sparring and pad work required similar VO? (43-41 ml·kg?1·min?1, respectively), which corresponds to ~70%VO?peak. These results underline the importance of a minimum of aerobic fitness for boxers and draw some guidelines for the intensity of training.     

Effects of olive leaf polyphenols against H₂O₂ toxicity in insulin secreting β-cells

In pancreatic β-cells, although H?O? is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H?O? (0.035 mM) for 45 min. H?O? alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H?O?-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H?O?-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.     

Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H₂O₂

Oxidative stress is a threat to mammalian cells. To better understand the molecular response and mechanism underlying oxidative stress, we applied two‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption ionization time‐of‐fight mass spectrometry analysis to identify differential nuclear protein profiling of mouse fibroblast NIH‐3T3 cells exposed to mild‐H₂O₂. Thirteen differentially expressed proteins were identified by MS and two of them were further validated by Western blot. The results revealed that exposure to mild‐H₂O₂ for 12 h cause up‐regulated expression of DJ‐1, glutathione S‐transferase P 1, DNA ligase I, dynamin 2, nucleophosmin, and down‐regulated expression of nucleoside diphosphate kinase A, enolase‐α, barrier‐to‐autointegration factor 1, metastasis associated protein 1, glycosytransferase‐like domain containing protein 1, synaptonemal complex protein 1, alpha‐centractin, bromodomain, and PHD finger containing 1 (BRPF1). Most of the identified proteins are supported as nuclear proteins localized by previous research. The findings may provide some clues to elucidate cell responses to H₂O₂ and the potential mechanism underlying protection against oxidative stress in fibroblast cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts

Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 μM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

ABA controls H₂O₂ accumulation through the induction of OsCATB in rice leaves under water stress

The production of both ABA and H?O? is induced by drought and can act as signals under stress conditions. We investigated the relationships between ABA, H?O? and catalase (CAT) in rice leaves when rice seedlings were treated with polyethylene glycol as water stress treatment. As a key gene in ABA biosynthesis, OsNCED3 was significantly induced in rice by water stress treatment and such induction preceded the rapid increase in ABA. Water stress inhibited the expression of CATA and CATC but substantially enhanced the expression of CATB. Exogenously applied ABA promoted the expression of CATB also and inhibited the expression of CATC in a concentration-dependent manner. When ABA production was inhibited by using ABA biosynthesis inhibitors nordihydroguaiaretic acid and tungstate, expression of CATB was also subdued while CATC was enhanced under the water stress. Accumulation of H?O? was also reduced when endogenous ABA production was inhibited and showed a correlation with the total activity of catalases. Our results suggest that water stress-induced ABA prevents the excessive accumulation of H?O?, through the induction of the expression of CATB gene during water stress.     

The soluble proteome of tobacco Bright Yellow-2 cells undergoing H₂O₂-induced programmed cell death

Plant programmed cell death (PCD) is a genetically controlled process that plays an important role in development and stress responses. Reactive oxygen species (ROS) are key inducers of PCD. The addition of 50 mM H?O? to tobacco Bright Yellow-2 (TBY-2) cell cultures induces PCD. A comparative proteomic analysis of TBY-2 cells treated with 50 mM H?O? for 30 min and 3 h was performed. The results showed early down-regulation of several elements in the cellular redox hub and inhibition of the protein repair-degradation system. The expression patterns of proteins involved in the homeostatic response, in particular those associated with metabolism, were consistently altered. The changes in abundance of several cytoskeleton proteins confirmed the active role of the cytoskeleton in PCD signalling. Cells undergoing H?O?-induced PCD fail to cope with oxidative stress. The antioxidant defence system and the anti-PCD signalling cascades are inhibited. This promotes a genetically programmed cell suicide pathway. Fifteen differentially expressed proteins showed an expression pattern similar to that previously observed in TBY-2 cells undergoing heat shock-induced PCD. The possibility that these proteins are part of a core complex required for PCD induction is discussed.     

The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.     

Adenosine A₂A and A₂B receptors are both required for adenosine A₁ receptor-mediated cardioprotection

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.     

Induction of apoptosis in non-small cell lung carcinoma A549 cells by PGD₂ metabolite, 15d-PGJ₂

PGD2 (prostaglandin D2) is a mediator in various pathophysiological processes, including inflammation and tumorigenesis. PGD2 can be converted into active metabolites and is known to activate two distinct receptors, DP (PGD2 receptor) and CRTH2/DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). In the past, PGD2 was thought to be involved principally in the process of inflammation. However, in recent years, several studies have shown that PGD2 has anti-proliferative ability against tumorigenesis and can induce cellular apoptosis via activation of the caspase-dependent pathway in human colorectal cancer cells, leukaemia cells and eosinophils. In the lung, where PGD2 is highly released when sensitized mast cells are challenged with allergen, the mechanism of PGD2-induced apoptosis is unclear. In the present study, A549 cells, a type of NSCLC (non-small cell lung carcinoma), were treated with PGD2 under various conditions, including while blocking DP and CRTH2/DP2 with the selective antagonists BWA868C and ramatroban respectively. We report here that PGD2 induces A549 cell death through the intrinsic apoptotic pathway, although the process does not appear to involve either DP or CRTH2/DP2. Similar results were also found with H2199 cells, another type of NSCLC. We found that PGD2 metabolites induce apoptosis effectively and that 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2) is a likely candidate for the principal apoptotic inducer in PGD2-induced apoptosis in NSCLC A549 cells.     

H₂O₂ stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4

In fission yeast, the stress-activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H(2)O(2) but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H(2)O(2), resulting in the full-length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4.