Hemoproteins affect H(2)O(2) removal from rat tissues

The capacity of rat liver homogenates and mitochondria to remove H(2)O(2) was determined by comparing their ability to slow fluorescence generated by a H(2)O(2) 'detector' with that of desferrioxamine solutions. H(2)O(2) was produced by glucose oxidase-catalysed glucose oxidation. The capacity to remove H(2)O(2) was expressed as equivalent concentration of desferrioxamine. The method showed changes in the capacity of H(2)O(2) removal after treatment with ter-butylhydroperoxide or glutathione. The H(2)O(2) removal capacity of homogenates and mitochondria from rat liver, heart, and skeletal muscle was compared with their overall antioxidant capacity. For homogenates, the order of both antioxidant and H(2)O(2) removal capacities was liver>heart>muscle. For mitochondria, the order of the antioxidant capacities mirrored that of the homogenates, while the order of the H(2)O(2) removal capacities was heart>muscle>liver. Because H(2)O(2) removal is not only due to H(2)O(2)-metabolizing enzymes, but also to hemoproteins that convert H(2)O(2) into more reactive radicals via Fenton reaction, the higher concentration of cytochromes in mitochondria of cardiac and skeletal muscles can explain the above discrepancy. A higher H(2)O(2) removal capacity was found to be associated with a higher rate of H(2)O(2) release by mitochondria, indicating that the order of H(2)O(2) release rate mirrors that of H(2)O(2) production rate. We suggest that the different capacities of the mitochondria from the three tissues to produce reactive oxygen species are due to differences in the concentration of respiratory mitochondrial chain components in the reduced form.     

Differential behavior of sPLA2-V and sPLA2-X in human neutrophils

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.     

Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes

Increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) promote cytosolic phospholipase A(2) (cPLA(2)) translocation to intracellular membranes. The specific membranes to which cPLA(2) translocates and the [Ca(2+)](i) signals required were investigated. Plasmids of EGFP fused to full-length cPLA(2) (EGFP-FL) or to the cPLA(2) C2 domain (EGFP-C2) were used in Ca(2+)/EGFP imaging experiments of cells treated with [Ca(2+)](i)-mobilizing agonists. EGFP-FL and -C2 translocated to Golgi in response to sustained [Ca(2+)](i) greater than approximately 100-125 nm and to Golgi, ER, and perinuclear membranes (PNM) at [Ca(2+)](i) greater than approximately 210-280 nm. In response to short duration [Ca(2+)](i) transients, EGFP-C2 translocated to Golgi, ER, and PNM, but EGFP-FL translocation was restricted to Golgi. However, EGFP-FL translocated to Golgi, ER, and PNM in response to long duration transients. In response to declining [Ca(2+)](i), EGFP-C2 readily dissociated from Golgi, but EGFP-FL dissociation was delayed. Agonist-induced arachidonic acid release was proportional to the [Ca(2+)](i) and to the extent of cPLA(2) translocation. In summary, we find that the differential translocation of cPLA(2) to Golgi or to ER and PNM is a function of [Ca(2+)](i) amplitude and duration. These results suggest that the cPLA(2) C2 domain regulates differential, Ca(2+)-dependent membrane targeting and that the catalytic domain regulates both the rate of translocation and enzyme residence.     

H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H(2)O(2)) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K(+) (K(V)) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H(2)O(2) increases K(+) current in coronary artery smooth muscle cells. H(2)O(2) increased K(+) current in a concentration-dependent manner (increases of 14 +/- 3 and 43 +/- 4% at 0 mV with 1 and 10 mM H(2)O(2), respectively). H(2)O(2) increased a conductance that was half-activated at -18 +/- 1 mV and half-inactivated at -36 +/- 2 mV. H(2)O(2) increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H(2)O(2) on K(+) current and significantly shifted the voltage of half-activation to -10 +/- 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H(2)O(2) to increase K(+) current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca(2+)-activated K(+) channels, blocked the effect of H(2)O(2) to increase K(+) current. In contrast, 3 mM 4-AP completely blocked the effect of H(2)O(2) to increase K(+) current. These findings lead us to conclude that H(2)O(2) increases the activity of 4-AP-sensitive K(V) channels. Furthermore, our data support the idea that 4-AP-sensitive K(V) channels are redox sensitive and contribute to H(2)O(2)-induced coronary vasodilation.     

Cellular components that functionally interact with signaling phospholipase A(2)s

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.     

On the existence of a possible A2A-D2-β-Arrestin2 complex: A2A agonist modulation of D2 agonist-induced β-arrestin2 recruitment

Given that coactivation of adenosine A(2A) (A(2A)R) and dopamine D(2) (D(2)R) receptors results in the coaggregation, cointernalization, and codesensitization of the A(2A)R and D(2)R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A(2A)R agonist CGS21680 in A(2A)R-D(2)R-coexpressing cells to modulate the D(2)R agonist-induced recruitment of β-arrestin2 to the D(2)R by means of proximity-based bioluminescence resonance energy transfer (BRET(2)) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET(2) signal between β-arrestin2(RLuc) and D(2L)R(GFP2) upon D(2)R activation, by increasing the potency of the D(2)R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2(GFP2) and D(2L)R(YFP) as seen from the co-trafficking analysis. Furthermore, the A(2A)R agonist advanced the time for the increase in Akt phosphorylation obtained with the D(2)R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A(2A)R-D(2)R heteromers. Taken together, the results indicate that the antagonistic A(2A)R-D(2)R allosteric receptor-receptor interaction in A(2A)R-D(2)R heteromers favors β-arrestin2 recruitment to the D(2L)R protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.     

Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.     

P2Y₂ receptor activation decreases blood pressure and increases renal Na⁺ excretion

ATP and UTP are endogenous agonists of P2Y(2/4) receptors. To define the in vivo effects of P2Y(2) receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y(2/4) receptor agonist and UTP analog, in wild-type (WT) and P2Y(2) receptor knockout (P2Y(2)-/-) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS45973 (0.1 to 3 mg/kg body wt) dose-dependently decreased blood pressure in WT (maximum response -35 ± 2 mmHg) and to a similar extent in endothelial nitric oxide synthase knockout mice. By contrast, blood pressure increased in P2Y(2)-/- (maximum response +18 ± 1 mmHg) but returned to basal levels within 60 s. Continuous infusion of INS45973 (25 to 750 μg·min(-1)·kg(-1) body wt) dose-dependently increased urinary excretion of Na(+) in WT (maximum response +46 ± 15%) but reduced Na(+) excretion in P2Y(2)-/- (maximum responses of -45 ± 15%) mice. In renal clearance experiments, INS45973 did not affect glomerular filtration rate but lowered blood pressure and increased fractional excretion of fluid, Na(+), and K(+) in WT relative to P2Y(2)-/- mice. The blood pressure responses to INS45973 are consistent with P2Y(2) receptor-mediated NO-independent vasodilation and implicate responses to endothelium-derived hyperpolarizing factor, and P2Y(2) receptor-independent vasoconstriction, probably via activation of P2Y(4) receptors on smooth muscle. Systemic activation of P2Y(2) receptors thus lowers blood pressure and inhibits renal Na(+) reabsorption, effects suggesting the potential utility of P2Y(2) agonism in the treatment of hypertension.     

Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).     

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).     

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.     

Biphasic modulation of fatty acid synthase by hydrogen peroxide in Saccharomyces cerevisiae

Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150μM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit β. However, the exposure to a steady-state 50μM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.     

Dissimilatory Reduction of NO(2) to NH(4) and N(2)O by a Soil Citrobacter sp

Dissimilatory reduction of NO(2) to N(2)O and NH(4) by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N(2)O production by this mechanism. In batch cultures with defined media, NO(2) reduction to NH(4) was favored by high glucose and low NO(3) concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO(3) concentrations. With succinate as the energy source, little or no NO(2) was reduced to NH(4) but N(2)O was produced. Resting cell suspensions reduced NO(2) simultaneously to N(2)O and free extracellular NH(4). Chloramphenicol prevented the induction of N(2)O-producing activity. The K(m) for NO(2) reduction to N(2)O was estimated to be 0.9 mM NO(2), yet the apparent K(m) for overall NO(2) reduction was considerably lower, no greater than 0.04 mM NO(2). Activities for N(2)O and NH(4) production increased markedly after depletion of NO(3) from the media. Amendment with NO(3) inhibited N(2)O and NH(4) production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH(4) but not of N(2)O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N(2)O at rates equal to the wild type. These observations suggest that N(2)O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO(2) to NH(4).     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Divalent metals inhibit and lactose stimulates zinc transport across brush border membrane vesicles from piglets

Interactions between metals of similar coordination chemistry are of relevance to infant nutrition due to the highly variable metal:metal ratios found in formulas. Using ratios similar to those found in infant formulas, our objectives were to determine the effects of metals and of lactose and other saccharides on Zn(+2) transport across intestinal brush border membranes. Brush border membrane vesicles prepared from intestines of 5 preweaned piglets were used to determine whether Ca(+2), Mg(+2), Fe(+2), Cu(+2), Cd(+2), or Mn(+2) would antagonize Zn(+2) uptake. (65)Zn(+2) uptake by brush border membrane vesicles was measured over 20 min with metal concentrations constant, and at 1 min with increasing metal concentrations. Zn(+2) bound to the external surface of vesicles was removed with ethylenediamine-tetraacetic acid. Lactose induced Zn(+2) uptake to a greater extent than glucose polymer, whereas maltose, galactose, or galactose/glucose had no effect. Over 20 min, a 10:1 concentration of Fe(+2), Cd(+2), Cu(+2), and Mn(+2) lowered Zn(+2) uptake significantly (P < 0.05). Higher concentrations of divalent cation significantly lowered Zn(+2) (0.2 or 0.1 mM) uptake for all metals tested (P < 0.05), except for Mn(+2) (0.1 mM Zn(+2)). Inhibition constant determination quantified relative competitive potential with Mg(+2) < Ca(+2) < Mn(+2) < Fe(+2) < Zn(+2) < Cu(+2). Relative amounts of Ca(+2), Mg(+2), and Fe(+2) similar to those found in infant formulas reduced Zn(+2) uptake by at least 40%. Our data demonstrate that dietary minerals compete during brush border membrane transport, and may help explain antagonistic mineral interactions observed in vivo. Divalent metal concentrations and lactose content of milk affect zinc absorption in neonates and must be carefully considered in formula design.     

A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: evaluation in human polymorphonuclear leukocytes

There has been a long-standing need for sensitive and specific techniques for hydrogen peroxide (H(2)O(2)) measurement. We describe the development and application of a highly sensitive electrochemical sensor, utilizing a membrane-coated platinum microelectrode, suitable for real-time measurement of hydrogen peroxide generation and consumption in biochemical or cellular systems. This sensor provides high sensitivity enabling measurement of hydrogen peroxide down to 5-10 nM concentrations. We demonstrate that it can be used to measure the magnitude and time course of H(2)O(2) generation from the NADPH oxidase in leukocytes as well as the rate of H(2)O(2) degradation. After human polymorphonuclear leukocytes (PMNs) were activated by phorbol 12-myristate acetate, H(2)O(2) concentration increased with time and reached a peak concentration, from 5 to 15 microM in PMNs prepared from different individuals, within 3 to 8 min, then decreased slowly. The H(2)O(2) concentration in the solution is less than the total H(2)O(2) generation from the activated PMNs because a part of H(2)O(2) generated is decomposed. H(2)O(2) in solution, generated from the PMNs, was rapidly consumed after the activated PMNs were treated with 10 microM diphenylene iodonium (DPI). The rate of H(2)O(2) consumption was measured following the addition of exogenous H(2)O(2). The total production of H(2)O(2) from the activated PMNs was calculated from the measured H(2)O(2) concentration and the rate of H(2)O(2) consumption. This technique enables sensitive and continuous real-time measurement of H(2)O(2) concentration and total H(2)O(2) generation in cellular or enzyme systems without addition of any detection reagents.     

Interaction of bovine uterine luminal protein with interleukin-2 and the interleukin-2 receptor of T lymphocytes

Bovine uterine luminal proteins (ULP) collected on Day 17 of pregnancy were tested for inhibition of binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) of bovine (CLC) and human (HLC) T lymphocytes and for binding to IL-2. Additional experiments assessed IL-2 binding to the p55 alpha chain (Tac protein) of the IL-2R of HLC. High- and low-molecular weight (Mr) ULP components (H-ULP greater than 248,000 Mr and L-ULP 21,000 Mr, respectively) inhibited (p less than 0.05 and 0.01, respectively) the binding of 125I-IL-2 to the IL-2R of CLC, whereas only H-ULP inhibited (p less than 0.05) binding to the IL-2R (presumably, the p75 beta chain) of HLC. H-ULP failed (p greater than 0.05) to bind to the p55 alpha chain of the IL-2R of HLC. For IL-2 binding, L-ULP failed (p greater than 0.05) to bind 125I-IL-2 in short (2 h)-term and long (45 h)-term experiments, whereas binding was evident (p less than 0.05) for H-ULP at 2 h of incubation. For H-ULP, mean (+/- SEM) percentages for bound and unbound 125I-IL-2 were 70.1 +/- 11.4 and 29.9 +/- 11.4, respectively. Further purification of H-ULP yielded a component (1.76 x 10(6) Mr) that bound 11.7% of 125I-IL-2 and inhibited (p less than 0.01) thymidine uptake and binding of 125I-IL-2 to the IL-2R of CLC. H-ULP-mediated suppression of lymphocyte proliferation may result from blocking IL-2R recognition of IL-2 as well as binding to IL-2, whereas suppression by L-ULP may predominantly result from blocking IL-2R.