Nmol Of O2 Research Articles

Functionalization of decavanadate anion by coordination to cobalt(II): Binding to proteins, cytotoxicity, and water oxidation catalysis

A series of five decavanadates (V10) using a simple, one-pot synthesis, adhering to the model template: transition metal ion – decavanadate – ligands:(Hnicotinamide)2{[Co(H2O)3(nicotinamide)2]2[μ-V10O28]}.6H2O (1), {[Co(H2O)4(isonicotinamide)2]3}V10O28·4H2O (2), {[Co(H2O)4]2[Co(H2O)2(μ-pyrazinamide)2][μ-V10O28]}·4H2O (3) {[Co(H2O)4(μ-pyrazinamide)]3.V10O28}·4H2O (4), and (NH4)2{[Ni(H2O)4(2-hydroxyethylpyridine)]2}V10O28·2H2O (5) was synthesized. X-ray analysis reveals that 1 and 3 are decavanadato complexes, while 2, 4 and 5 are decavanadate complex salts. Moreover, 3 is the first example of a polymeric decavanadato complex, employing direct coordination with the metal center and the organic ligand, in toto. From the solution studies using 51V NMR spectroscopy, it was decoded that 1 and 3 stay stable in the model buffer solution and aqueous media. Binding to model proteins, cytotoxicity and water oxidation catalysis (WOC) was studied primarily for 1 and 3 and concluded that neither 1 nor 3 have an interaction with the model proteins thaumatin, lysozyme and proteinase K, because of the presence of the organic ligands in the Co(II) center, any further interplay with the proteins was blocked. Cytotoxicity studies reveal that 1 is 40% less toxic (0.05 mM) and 26% less toxic (0.1 mM) than the uncoordinated V10 with human cell lines A549 and HeLa respectively. In WOC, 1 performed superior activity, by evolving 143.37 nmol of O2 which is 700% (9-fold) increase than the uncoordinated V10.

Dysregulated Mitochondrial Respiration Occurs With Aging

Numerous metabolic changes occur in skeletal muscle with aging. At the crossroads of these changes lies the cellular energy highway, the mitochondrial reticulum. Aging is associated with altered patterns of mitochondrial dynamics and morphology, which are thought to be primarily driven by mitochondrial fragmentation. This fragmentation phenomenon may also lead to altered mitochondrial respiration with aging. We used a mouse model and measured parameters of respiration in muscle mitochondrial preparations to evaluate the hypothesis of aging‐related effects on metabolism and energy substrate partitioning. Mixed skeletal muscle of C57/Bl6 mice (n=32) was excised from single hind‐limbs of young males (YM) (n=8), old males (OM) (n=8), young females (YF) (n=8) and old females (OF) (n=8). Tissues were homogenized in an isotonic solution supplemented with 0.15% protease inhibitor. Mitochondria were isolated by differential centrifugation and their concentrations were determined by BCA analysis. Isolated mitochondria (0.1mg of mitochondria/mL of respiration medium) were then respired using a Clarke‐Type Electrode at 25°C with 100 nM of ADP and either 10 mM Pyruvate+ 2.5 mM Malate (P+M) or 40 uM Palmitoyl‐L‐Carnitine+ 1 mM Malate (PC+M) as substrates. Respiratory Control Ratios (RCR) were calculated as the quotient of State 3 (ADP‐stimulated respiration) and State 4 (recovery rate post ATP synthesis) respiratory rates (State 3/State 4) and compared between substrate and age. ADP to oxygen ratios (P:O) were calculated by dividing the amount of ADP added to the respiratory chamber by the oxygen consumption rate (nmol of O2/mg of mitochondria/minute) of state 3 respiration. RCR in both P+M and PC+M significantly decreased with aging [P+M (p<0.05): YM (4.1±0.9) vs. OM (2.7±1.1); YF (5.1±1.4) vs. OF (3.2±2)] [PC+M (p<0.05): YM (4.2±0.9) vs. OM (2.4±0.7); YF (3.4.±0.5) vs. OF (2.2±0.9)]. P:O in both P+M and PC+M did not significantly change with aging [P+M: (p=0.69): YM (2.9±0.3) vs. OM (2.9±0.2); YF: (p=0.07): (3±0.1) vs. OF (2.9±0.1)] [PC+M:(p=0.65); YM (2.9±0.4) vs. OM (2.9±0.3); (p=0.22): YF (2.9±0.2) vs. OF (2.7±0.3)]. While there were no significant changes in the P:O the age related decreases in the RCR were characterized by a lower capacity to respire derivatives of both CHO and FA. Decreased State 3 respiration and RCR typically corresponds to loss of respiratory capacity and loosening of coupling efficiency. Because exercise increases mitochondrial function and biogenesis it may be prudent to study older animals after exercise training. We plan to further investigate the structural changes in the mitochondrial reticulum and proteome that may be altering mitochondrial function in aging.

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1G93A motor neurons.

Angiotensin II–Dependent Hypertension Increases Na Transport-Related Oxygen Consumption by the Thick Ascending Limb

Renal medullary superoxide (O(2)(-)) increases in angiotensin (Ang) II-dependent hypertension. O(2)(-) increases thick ascending limb Na transport, but the effect of Ang II-dependent hypertension on the thick ascending limb is unknown. We hypothesized that Ang II-dependent hypertension increases thick ascending limb NaCl transport because of enhanced O(2)(-) production and increased protein kinase C (PKC) alpha activity. We measured the effect of Ang II-dependent hypertension on furosemide-sensitive oxygen consumption (a measure of Na transport), O(2)(-) production, and PKCalpha translocation (a measure of PKCalpha activity) in thick ascending limb suspensions. Ang II-dependent hypertension increased furosemide-sensitive oxygen consumption (26.2+/-1.0% versus 36.6+/-1.2% of total oxygen consumption; P<0.01). O(2)(-) was also increased (1.1+/-0.2 versus 3.2+/-0.5 nmol of O(2)(-)/min per milligram of protein; P<0.03) in thick ascending limbs. Unilateral renal infusion of Tempol decreased O(2)(-) (2.4+/-0.4 versus 1.2+/-0.2 nmol of O(2)(-)/min per milligram of protein; P<0.04) and furosemide-sensitive oxygen consumption (32.8+/-1.3% versus 24.0+/-2.1% of total oxygen consumption; P<0.01) in hypertensive rats. Tempol did not affect O(2)(-) or furosemide-sensitive oxygen consumption in normotensive controls and did not alter systolic blood pressure. Ang II-dependent hypertension increased PKCalpha translocation (5.7+/-0.3 versus 13.8+/-1.4 AU per milligram of protein; P<0.01). Unilateral renal infusion of Tempol reduced PKCalpha translocation (5.0+/-0.9 versus 10.4+/-2.6 AU per milligram of protein; P<0.04) in hypertensive rats. Unilateral renal infusion of the PKCalpha inhibitor Gö6976 reduced furosemide-sensitive oxygen consumption (37.4+/-1.5% versus 25.1+/-1.0% of total oxygen consumption; P<0.01) in hypertensive rats. We conclude that Ang II-dependent hypertension enhances thick ascending limb Na transport-related oxygen consumption by increasing O(2)(-) and PKCalpha activity.

Critical Role of the NAD(P)H Oxidase Subunit p47 phox for Left Ventricular Remodeling/Dysfunction and Survival After Myocardial Infarction

Accumulating evidence suggests a critical role of increased reactive oxygen species production for left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). An increased myocardial activity of the NAD(P)H oxidase, a major oxidant enzyme system, has been observed in human heart failure; however, the role of the NAD(P)H oxidase for LV remodeling and dysfunction after MI remains to be determined. MI was induced in wild-type (WT) mice (n=46) and mice lacking the cytosolic NAD(P)H oxidase component p47(phox) (p47(phox)-/- mice) (n=32). Infarct size was similar among the groups. NAD(P)H oxidase activity was markedly increased in remote LV myocardium of WT mice after MI as compared with sham-operated mice (83+/-8 versus 16.7+/-3.5 nmol of O(2)(-) x microg(-1) x min(-1); P<0.01) but not in p47(phox)-/- mice after MI (13.5+/-3.6 versus 15.5+/-3.5 nmol of O(2)(-) x microg(-1) x min(-1)), as assessed by electron-spin resonance spectroscopy using the spin probe CP-H. Furthermore, increased myocardial xanthine oxidase activity was observed in WT, but not in p47(phox)-/- mice after MI, suggesting NAD(P)H oxidase-dependent xanthine oxidase activation. Myocardial reactive oxygen species production was increased in WT mice, but not in p47(phox)-/- mice, after MI. LV cavity dilatation and dysfunction 4 weeks after MI were markedly attenuated in p47(phox)-/- mice as compared with WT mice, as assessed by echocardiography (LV end-diastolic diameter: 4.5+/-0.2 versus 6.3+/-0.3 mm, P<0.01; LV ejection fraction, 35.8+/-2.5 versus 22.6+/-4.4%, P<0.05). Furthermore, cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis were substantially reduced in p47(phox)-/- mice as compared with WT mice. Importantly, the survival rate was markedly higher in p47(phox)-/- mice as compared with WT mice after MI (72% versus 48%; P<0.05). These results suggest a pivotal role of NAD(P)H oxidase activation and its subunit p47(phox) for LV remodeling/dysfunction and survival after MI. The NAD(P)H oxidase system represents therefore a potential novel therapeutic target to prevent cardiac failure after MI.

Nitric oxide and superoxide radical production by human mononuclear leukocytes.

Human mononuclear cells (90% lymphocytes, 9% monocytes, and 1% polymorphonuclear leukocytes) produced spontaneously in resting state 0.11+/-0.01 nmol of nitric oxide (NO)/min/10(6) cells and 0.25+/-0.02 nmol of superoxide anion (O2-)/min/10(6) cells, as primary products. When these cells were stimulated with phorbol 12-myristate 13-acetate (PMA), the NO and O2- production increased by 82% and 204% to 0.25+/-0.02 nmol of NO/min/10(6) cells and 0.76+/-0.12 nmol of O2-/min/10(6) cells, respectively. Oxygen uptake reasonably accounted for the sum of the rates of NO and hydrogen peroxide (H2O2), the latter calculated as 0.5 O2- production, in nonstimulated and in PMA-stimulated cells. H2O2 and peroxynitrite formation were detected and measured as secondary products of the primary products O2- and NO. An original assay to determine H2O2 steady-state concentration and production rates is described. The determined production rates of the involved reactive species are in good agreement with known chemical equations. It is apparent that NO and O2- production by human mononuclear cells may constitute the basis of intercellular signaling and cell toxicity.

Superoxide anion production in influenza protein-activated NADPH oxidase of human polymorphonuclear leukocytes.

There are conflicting reports regarding superoxide anion (O2-) production by human polymorphonuclear leukocytes (PMNL) that have been activated by influenza virus. In the present study, the output of O2- was determined by measuring superoxide dismutase-inhibitable cytochrome c reduction. Incubation of PMNL with purified influenza matrix (M) protein, neuraminidase (NA), or hemagglutinin (HA) enhanced the production of O2-: 4.93 nmol of O2-/4 x 10(5) cells/15 min was produced with M protein, 5.20 with NA, and 6.89 with HA. These values were significantly higher (P < .05) than that for untreated PMNL (1.51). Both nonglycosylated and glycosylated proteins had the potential to generate O2- in human PMNL. Neither the hemagglutinating activity of HA nor the enzymatic activity of NA were necessary for viral protein activation of PMNL.

Extracellular production of singlet oxygen by stimulated macrophages quantified using 9,10-diphenylanthracene and perylene in a polystyrene film

The extracellular production of singlet oxygen (O2(1 delta g)) by stimulated macrophages was measured using a modification of our quantitative method initially developed to measure the intracellular production of O2(1 delta g) by neutrophils (Steinbeck, M. J., Khan, A. U., and Karnovsky, M. J. (1992) J. Biol. Chem. 267, 13425-13433). Glass coverslips were coated with the specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA) and perylene, which is an internal standard, in a methylene chloride solution containing 0.3 mg/ml polystyrene. On evaporation, the polystyrene formed an even coating of DPA and perylene over the surface of a glass coverslip (PDP film). Unstimulated macrophages or macrophages stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) were then added to the PDP film in a darkened room and incubated at 37 degrees C for 30 min in a humidified 5% CO2 atmosphere. Both unstimulated and stimulated cells adhered to the PDP film in approximately equivalent numbers. Only stimulated cells produced measurable amounts of O2(1 delta g) in a dose-dependent response to either PMA or fMLP. The production of O2(1 delta g) by macrophages stimulated with PMA was maximal in response to 25 ng, 17.8 +/- 1.3 nmol of O2(1 delta g)/approximately 1.00 x 10(6) cells. The maximal response for fMLP was at a concentration of 1 microM, 18.4 +/- 1.0 nmol of O2(1 delta g)/approximately 1.00 x 10(6) cells. The specific detection of O2(1 delta g) by this method was confirmed by thermally releasing O2(1 delta g) from the DPA-O2(1 delta g) reaction product, DPA-endoperoxide, regenerating the original DPA compound. Production of O2(1 delta g) by the stimulated cells was inhibited 80-89% by the addition of 60-120 micrograms of superoxide dismutase, an enzyme that converts superoxide to hydrogen peroxide and ground state molecular oxygen or 79-84% with the addition of 2 mM histidine, an avid quencher of O2(1 delta g). Neither of these additions interfered with adhesion of the cells to the PDP film. The ability of superoxide dismutase to inhibit the production of O2(1 delta g) suggested that O2(1 delta g) was produced via a superoxide-dependent route. The ability of an oxidase to produce O2(1 delta g) secondary to superoxide production was substantiated further using a xanthine oxidase-acetaldehyde system. Purified xanthine oxidase produced both superoxide and O2(1 delta g), and their production was inhibited by the addition of superoxide dismutase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of leucocytes on the hypoosmotic swelling test of human sperm.

The hypoosmotic swelling test was used to evaluate the size of physiologically competent sperm after their exposure to the influence of leucocyte-derived superoxide anions. In sperm suspensions, preincubated in the presence of 50 x 10(4)mL-1 leucocytes capable of producing 0.93 +/- 0.63 nmol of O2- (range 0.21-2.03 nmol) over 30 min at 37 degrees C, a significant decrease in the percentage of swollen sperm compared to that in the control sperm suspensions without added leucocytes was found (p < .001, n = 46). Addition of phorbol myristate acetate, an activator of the release of O2-, further diminished the ability of hypoosmotic sperm tail swelling (p < .001), while superoxide dismutase inhibited this effect. This study suggests that even very low amounts of superoxide anions may influence the functional integrity of sperm. Therefore, any source of superoxide anion production should be eliminated and/or inhibited in sperm suspensions used in assisted reproduction techniques.

In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease.

Epstein-Barr virus-transformed lymphocytes generate superoxide in response to various agonists in an enzymatic reaction similar to that which occurs in stimulated phagocytes. We generated transformed B lymphoblast cell lines from controls, from four patients with p47-phox-deficient chronic granulomatous disease, and from three parents. The cells from controls and from the parents generated 7.0-35 nmol of O2-/10(7) cells per 30 min in response to phorbol myristate acetate. None of the patient cell lines generated any detectable superoxide. Both p47-phox and p67-phox were detected by immunoblot in the cytosol of control and parent cell lines and, as in neutrophils, these proteins had affinity for GTP-agarose. The patients' cell lines contained no detectable p47-phox by immunoblot. mRNA for both cytosolic proteins was detected in all cell lines. We generated cDNA and obtained multiple clones from two patients by polymerase chain reaction. One patient was a compound heterozygote with each allele resulting in an early stop codon. Clones derived from the other patient demonstrated only a GT deletion at base 75. The cDNA for p47-phox was inserted into an EBV-expression vector and stably transfected cell lines were obtained using hygromycin B selection. Transfected cell lines from a p47-phox-deficient patient generated normal levels of superoxide and had readily detectable cytosolic p47-phox. Thus, B lymphoblasts provide an excellent model system for studies of the NADPH oxidase, for expression of functional recombinant forms of oxidase components, and for initial experimental approaches to genetic reconstitution in CGD.

Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.

To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo activation of peripheral blood polymorphonuclear neutrophils by gamma interferon results in enhanced fungal killing

The effect of in vivo administration of murine recombinant gamma interferon (IFN) on the fungicidal activity of murine peripheral blood polymorphonuclear neutrophils (PB-PMNs) was studied. Mice were injected intramuscularly with 250, 2,500, 25,000 or 250,000 U of IFN 5 h before collection of peripheral blood. Purified PB-PMNs were cocultured in vitro with Blastomyces dermatitidis yeast cells for 2 h. PB-PMNs from untreated mice killed 44.5 +/- 12.5% of the fungal inoculum, whereas PB-PMNs from mice treated with 25,000 or 250,000 U of IFN showed significantly enhanced in vitro killing (68.0 +/- 9.4% [P less than 0.005] and 72.3 +/- 1.1% [P less than 0.001], respectively). Treatment with 250 or 2,500 U of IFN or 25,000 U of heated (100 degrees C, 15 min) IFN had no effect. The IFN-induced activation of PB-PMNs was transitory. Significant enhancement of PB-PMN killing activity occurred 1, 2, or 5 h after in vivo IFN administration, but no enhancement was observed 16 or 24 h after IFN treatment. Enhanced fungicidal activity by PB-PMNs from mice treated for 5 h with 25,000 U of IFN correlated with an increased release of superoxide anion (O2-) in vitro after stimulation of PB-PMNs with phorbol ester; normal PB-PMNs and IFN-activated PB-PMNs, respectively, produced 2.2 +/- 2.5 and 23.5 +/- 4.8 nmol of O2- per 10(6) PB-PMNs per 30 min (P less than 0.005). The exogenous addition of compounds that antagonize or inhibit the formation of oxygen radicals (superoxide dismutase, catalase, dimethyl sulfoxide, or sodium azide) significantly inhibited fungal killing by both normal and IFN-activated PB-PMNs. In addition to the enhanced microbicidal activity and superoxide generation demonstrated in vitro with constant cell numbers, there was a transient leukocytosis (particularly neutrophilia) in peripheral blood at doses of IFN and at times after IFN administration where enhanced activity was also demonstrated. In summary, our results indicate that PB-PMNs can be activated in vivo for enhanced killing of a fungal target. The enhanced killing capacity of IFN-activated PB-PMNs is due at least in part to the enhancement of oxidative killing mechanisms.

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.

Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli.

We compared the degree to which Escherichia coli phase variants which do (T1P+ E. coli) or do not (T1P- E. coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity. Unopsonized T1P+ E. coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E. coli. In the presence of serum opsonins, T1P+ E. coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E. coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min. Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity. The activity stimulated by either T1P+ E. coli or T1P-latex was susceptible to inhibition by cytochalasin B. Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN. These data indicate that T1P+ E. coli stimulate PMN oxidative metabolism more effectively than do T1P- E. coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E. coli. Furthermore, T1P-latex faithfully mimics the ability of T1P+ E. coli to stimulate PMN oxidative metabolism. Such particles may be useful in further analyses of cellular responses to T1P+ E. coli.

Retinoids stimulate the release of superoxide by neutrophils and change their morphology.

All-trans-retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 +/- 14 SD and 53 +/- 5 SD nmol of O2-/min/10(7) cells, respectively. Superoxide release by unstimulated cells was negligible. All-trans-retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis-unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870-7877) are discussed in light of possible mechanisms.

Oxidative metabolism of neonatal and adult rabbit lung macrophages stimulated with opsonized group B streptococci.

We compared the oxidative metabolism of alveolar macrophages (AM) from adult and neonatal (1- and 7-day-old) rabbits before and after their in vitro exposure to type Ia group B streptococci (GBS) opsonized with immune rabbit serum. Nonstimulated AM from 1-day-old, 7-day-old, or adult rabbits consumed O2 at a rate of 17 to 20 nmol/10(6) AM per 10 min under basal conditions and released minimal amounts of superoxide (O2-) into the medium. Approximately 80% of this basal respiration was of mitochondrial origin, based on its inhibition by NaCN. Exposure to GBS opsonized with immune rabbit serum stimulated O2 consumption approximately half as effectively in the neonatal AM as in the adult AM. Little O2- was released into the medium unless the cells were pretreated with dihydrocytochalasin B. Under such conditions, 1-day-old, 7-day-old, and adult AM released 3.6, 5.3, and 13.9 nmol of O2-/10(6) AM per 10 min, respectively. The uptake of opsonized GBS by 1-day-old AM was not affected by 1 mM NaCN, whereas phagocytosis by adult AM was substantially reduced under these conditions. Overall, our findings suggest that neonatal AM have less-well-developed postphagocytic oxidative metabolic responses and release less superoxide after exposure to opsonized GBS than do adult AM. They also demonstrate that the energy for phagocytosis is derived principally from mitochondrial metabolism in adult AM but not in neonatal AM. We conclude that metabolic differences between neonatal and adult AM may contribute to neonatal pulmonary susceptibility to GBS infections and account, in part, for the ability of GBS to succeed as neonatal pulmonary pathogens.

Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites

Acetate dominated the extracellular pool of volatile fatty acids (VFAs) in the hindgut fluid of Reticulitermes flavipes, Zootermopsis angusticollis, and Incisitermes schwarzi, where it occurred at concentrations of 57.9 to 80.6 mM and accounted for 94 to 98 mol% of all VFAs. Small amounts of C(3) to C(5) VFAs were also observed. Acetate was also the major VFA in hindgut homogenates of Schedorhinotermes lamanianus, Prorhinotermes simplex, Coptotermes formosanus, and Nasutitermes corniger. Estimates of in situ acetogenesis by the hindgut microbiota of R. flavipes (20.2 to 43.3 nmol . termite . h) revealed that this activity could support 77 to 100% of the respiratory requirements of the termite (51.6 to 63.6 nmol of O(2) . termite . h). This conclusion was buttressed by the demonstration of acetate in R. flavipes hemolymph (at 9.0 to 11.6 mM), but not in feces, and by the ability of termite tissues to readily oxidize acetate to CO(2). About 85% of the acetate produced by the hindgut microbiota was derived from cellulose C; the remainder was derived from hemicellulose C. Selective removal of major groups of microbes from the hindgut of R. flavipes indicated that protozoa were primarily responsible for acetogenesis but that bacteria also functioned in this capacity. H(2) and CH(4) were evolved by R. flavipes (usually about 0.4 nmol . termite . h), but these compounds represented a minor fate of electrons derived from wood dissimilation within R. flavipes. A working model is proposed for symbiotic wood polysaccharide degradation in R. flavipes, and the possible roles of individual gut microbes, including CO(2)-reducing acetogenic bacteria, are discussed.

Cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils.

Arachidonate stimulates the production of large quantities of superoxide by human neutrophils: 93.8 +/- 12.5 S.D. nmol of O2(-)/min/10(7) cells. This rate is comparable to that observed with the most effective neutrophil-stimulating agents previously reported. Other cis-unsaturated fatty acids are also capable of eliciting this response, the order of effectiveness being: arachidonate greater than gamma-linolenate greater than linoleate greater than oleate. Linolelaidate, myristate, and palmitate are ineffective. These data are discussed in relation to recent reports concerning the oxidation of arachidonic acid by human neutrophils and by a cell-free system that generates superoxide and hydrogen peroxide.

Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria

Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2- -dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.