Excited state intramolecular proton transfer in 3-hydroxy flavone and 5-hydroxy flavone: A DFT based comparative study

Potential energy (PE) curves for the intramolecular proton transfer in the ground (GSIPT) and excited (ESIPT) states of 3-hydroxy-flavone (3HF) and 5-hydroxy-flavone (5HF) were studied using DFT/B3LYP (6-31G (d,p)) and TD-DFT/B3LYP (6-31G (d,p)) level of theory respectively. Our calculations suggest the non-viability of ground state intramolecular proton transfer for both the compounds. Calculated PE curves of 3HF for the ground and excited singlet states proton transfer process explain its four state laser diagram. Excited states PE calculations support the ESIPT process to both 5HF and 3HF. The difference in ESIPT emission process of 3HF and 5HF have been explained in terms of HOMO and LUMO electron distribution of the enol and keto tautomer of these two compounds.     

The use of in vivo microscopy to image the cnidarian stress response in three octocoral species

Energy‐converting symbionts and organelles may be central to the cnidarian stress response. Stress may damage photochemistry in the endosymbiotic dinoflagellate Symbiodinium spp., leaving photosystems I and II relatively reduced and increasing reactive oxygen species (ROS). Alternatively, ROS may emanate from mitochondria of either the host or the symbiont, or both. These sources of ROS can be distinguished by using microscopy to examine the effects of light on stressed cnidarians incubated in the dark with a fluorescent, ROS‐detecting probe. Experiments were carried out with three species of alcyonacean octocoral, Phenganax parrini, Sarcothelia sp., and Sympodium sp. After incubation of colonies for 1 h at elevated temperature, imaging and illumination (excitation 450–490 nm, emission 515–565 nm) were begun simultaneously. Formation of ROS largely corresponded to the onset of illumination. On the other hand, chlorophyll fluorescence (excitation 530–580 nm, emission 620–690 nm) did not conform to this pattern. This difference is consistent with the expected rates of reaction. Remarkably, treatment with the inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) resulted in dramatically higher levels of light‐induced ROS. Chlorophyll fluorescence was higher in the DCMU treatment but not significantly so. By controlling for variation between individual symbionts, however, DCMU produced significantly greater levels of chlorophyll fluorescence, indicating the expected greater reduction of photosystem II. A brief exposure to light and thermal stress produced a similar effect in all three species. In addition to ROS being initiated by light, these results indicate that: (1) a brief period of stress shifts photosystem redox state toward reduction, (2) photosystem II can donate electrons to oxygen when blocked with DCMU, and (3) chlorophyll fluorescence is highly variable among individuals of Symbiodinium. Imaging of individual symbionts in hospite thus provides a powerful method for understanding the initial steps of the cnidarian stress response.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells

Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 μM PE to induce hypertrophy after 24 and 48 h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4 h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4 h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48 h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells.     

A purified green barley extract with modulatory properties upon TNF alpha and ROS released by human specialised cells isolated from RA patients

Based on the finding that reactive oxygen species (ROS) play important roles in mediating proinflammatory cytokine production (e.g. TNF alpha) and that many plant extracts contain substances with antioxidant properties, we examined the antiinflammatory action of a green barley extract, commercially available as "Natural SOD". The results obtained in this study demonstrate that the antiinflammatory properties of "Natural SOD" due to its micromolecular substances, able to scavenge ROS and to down-regulate TNF alpha production, main inflammation mediators produced by specialised cells from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA). We prepared and tested a purified green barley extract (PE) containing micromolecular substances under 1 kDa, able to inhibit TNF alpha releasing--measured by an bioassay--from LPS stimulated human mononuclear cells (MNC) isolated both from PB and SF of RA patients. Luminol-dependent chemiluminescence has been used to measure the scavenging activity of PE on ROS releasing from activated neutrophils isolated from PB of RA patients. PE, containing high concentrations of substances with antioxidant and antiinflammatory properties, could be a more efficient natural drug for human use than "Natural SOD" in the treatment of RA patients.     

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes

The activities of enzymes involved in lipid metabolism—phospholipase A₂ (PLA₂) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA₂ activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA₂ activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA₂ activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA₂ activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA₂ activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.     

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Excited state intramolecular proton transfer in 3-hydroxychromone: a DFT-based computational study

Potential energy (PE) curves for intramolecular proton transfer in the ground (GSIPT) and intramolecular proton transfer in the excited (ESIPT) states of 3-hydroxychromone (3HC) have been studied using DFT-B3LYP/6-31G(d,p) and TD-DFT/6-31G(d,p) level of theory, respectively. Our calculations suggest the non-viability of GSIPT in 3HC. Excited states PE calculations show the existence of ESIPT process in 3HC. ESIPT in 3HC has also been explained in terms of HOMO and LUMO electron densities of the enol and keto tautomers of 3HC.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

One- and two-photon induced QD-based energy transfer and the influence of multiple QD excitations.

Due to the increased use of quantum dots (QDs) in diverse laser microscopies, it is interesting to study the excitation pump power and excitation wavelength dependence of QD-based energy transfer (ET) processes. The ET in QD conjugates with phthalocyanines (Pcs) was studied with femtosecond time-resolved pump-probe spectroscopy upon one- and two-photon excitation. At the used excitation wavelengths only the QDs are excited and become the energy donors. Due to the matched spectral overlap of QD photoluminescence and Pc absorption, the ET occurs on a picosecond time scale. The ET process shows strong pump power dependence whereby an increase in excitation power results in multiple QD excitations and in shorter excited state lifetimes on the QDs due to Auger relaxation. As a result, high excitation pump power leads also to an accelerated ET to the acceptor molecules from the initially multiply excited states of the QDs. Excited state quenching studies as function of pump power suggest that ET occurs mainly from the lowest one-exciton state (n = 1) and only to a minor extent from the multiply excited states (n > 1). For the short-lived, multiply excited states the ET competes inefficiently with Auger recombinations and energy transfer efficiencies of phi(ET)(n=1>) approximately 20%, phi(ET)(n=2>) approximately 7%, phi(ET)(n=3>) < or = 2% were obtained. Also after two-photon excitation the ET efficiency is highest from the one-exciton state. The experimentally determined ET efficiencies were compared with theoretical ET efficiencies upon multiple excitations. In both cases the ET efficiency decreases with the increase in excitation pump power.     

Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, beta-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2*-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc'). The O2*- generation and Asc formation were detected by O2*--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2*- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2*- and Asc*. Addition of GSH completely inhibited the PEA-dependent production of O2*- and Asc* in Hb solution. We propose that the O2*- generation and Asc* formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

Regulation of excitation energy transfer in organisms containing phycobilins

The mechanism of excitation energy redistribution (state transition) in organisms containing phycobilins is reviewed. Recent measurements using time-resolved fluorescence spectroscopy in the picosecond range confirm that the state transition in cyanobacteria and red algae is controlled by changes in the kinetics of energy transfer from PS 2 to PS 1 (spillover) rather than by physical dislocation of the phycobilisome and reassociation between the two photosystems (mobile antenna model). Contrary to the analogous situation in higher plants, there is no compelling evidence for the involvement of a protein phosphorylation event in the rapid time range of the state transition, but a variety of data indicate that a membrane conformational change occurs that might change the relative distance between, and/or orientation of the two photosystems within the thylakoid. The state transition is most probably initiated by the redox state of the intersystem electron transport chain, and the conversion to state 1 is driven by coupled PS1 cyclic electron transport. The cryptomonads also undergo wavelength dependent changes in excitation energy distribution by a mechanism very similar to that observed in the red algae and cyanobacteria. However, the changes in energy distribution in this group are most likely related to a photoprotection mechanism for PS2 rather than to a state transition.Abbreviations APC allophycocyanin - EF exoplasmic face - PE phycoerythrin - PC phycocyanin - PF protoplasmic face - LHC light harvesting chlorophyll a/b protein - PBS phycobilisome - LD linear dichroism - RC reaction center     

Peptide models of the helical hydrophobic transmembrane segments of membrane proteins: interactions of acetyl-K2-(LA)12-K2-amide with phosphatidylethanolamine bilayer membranes

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, acetyl-Lys(2)-(Leu-Ala)(12)-Lys(2)-amide [(LA)(12)], and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mole fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the protein/lipid ratio. However, the fractional contribution of this component to the total enthalpy changes decreases with increases in peptide concentration, and this component completely disappears at higher protein mole fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher protein concentrations. These two components of the DSC endotherm have been assigned to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in (LA)(12) concentration, the magnitude of this downward shift is progressively greater as the length of the PE hydrocarbon chain decreases. As well, mixtures of (LA)(12) with the longer chain PEs exhibit unusual biomodal enthalpy variations, suggesting peptide immiscibility in thicker gel state bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to zero even at high peptide concentrations, indicating that (LA)(12) attenuates but does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our FTIR spectroscopic data indicate that (LA)(12) remains in a predominantly alpha-helical conformation in liquid-crystalline PE bilayers of various hydrophobic thickness but that the helical conformation is altered in gel-state PE bilayers generally, probably due to peptide lateral aggregation. These data also suggest that (LA)(12) significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states, relatively independently of lipid hydrocarbon chain length. Many aspects of PE/(LA)(12) interactions exhibit a different dependence on the hydrophobic thickness of the host bilayer than was observed in our previous study of (LA)(12)-phosphatidylcholine (PC) model membranes [Zhang et al. (1995) Biochemistry 34, 2362-2371]. The differing effects of (LA)(12) incorporation on PE and PC bilayers is ascribed primarily to the much stronger lipid polar headgroup interactions characteristic of the former system. Finally, the considerable differences observed in the behavior of (LA)(12) and the related polyleucine-based peptide P(24) in both PC and PE bilayers indicate that the structure of the hydrophobic core of alpha-helical transmembrane peptides can affect their conformational plasticity and state of aggregation and thus the nature of their interactions with different phospholipid bilayers.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

A comparison of arteries and veins in oxidative stress: producers, destroyers, function, and disease

Reactive oxygen species (ROS) are by-products of oxygen metabolism, normally present in low levels inside cells, where they participate in signaling processes. The delicate balance in the continuous cycle of ROS generation and inactivation is maintained by enzymatic and nonenzymatic endogenous systems. Overwhelming production of ROS (by such sources as the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidase, or uncoupled nitric oxide synthase), when inadequately counteracted by destruction through antioxidant systems (such as superoxide dismutase or catalase), leads to a prooxidant state also known as oxidative stress. Increased levels of ROS and markers of oxidative stress have been consistently found in such cardiovascular diseases as atherosclerosis or hypertension, although controversy still exists over the pathophysiological role of oxidative stress in these conditions. ROS can modulate vascular function either by direct oxidative damage or by activating cellular signaling pathways that lead to abnormal contractile, inflammatory, proliferative, or remodeling properties of the blood vessel. Most current research focuses on these processes in arteries, leaving veins, "the other side" of vascular biology, in obscurity. Veins are different structurally and functionally from arteries. Equipped with a smaller smooth muscle layer compared to arteries, but being able to accommodate 70% of the circulating blood volume, veins can modulate cardiovascular homeostasis and contribute significantly to hypertension pathogenesis. Although the reports on the quantitative differences in ROS production in veins compared to arteries had conflicting results, there is a clear qualitative difference in ROS metabolism and utilization between the two vessel types. This review will compare and contrast the current knowledge of ROS metabolism in arteries versus veins in both physiological and pathophysiological conditions. Our understanding of the mechanisms underlying vascular diseases would greatly benefit from a more thorough exploration of the role of veins and venous oxidative stress.     

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

Elevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨ_m) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨ_m and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance.     

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

Elevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨ_m) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨ_m and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.