Nitrogen uptake and assimilation in Enteromorpha intestinalis (L.) Link (Chlorophyta): using N to determine preference during simultaneous pulses of nitrate and ammonium

We investigated the ability of Enteromorpha intestinalis (L.) Link to take up pulses of different species of nitrogen simultaneously, as this would be an important mechanism to enhance bloom ability in estuaries. Uptake rates and preference for NH₄⁺ or NO₃⁻ following 1, 3, 6, 9, 12 or 24 h of exposure to either ¹⁵NH₄NO₃ or NH₄¹⁵NO₃ were determined by disappearance of N from the medium. Differences in assimilation rates for NH₄⁺ or NO₃⁻ were quantified by the accumulation of NH₄⁺, NO₃⁻, and atom % ¹⁵N in the algal tissue. NH₄⁺ concentration was reduced more quickly than water NO₃⁻ concentration. Water column NH₄⁺ concentration after the longest time interval was reduced from 300 to 50 μM. Water NO₃⁻ was reduced from 300 to 150 μM. The presence of ¹⁵N or ¹⁴N had no effect on uptake of either NH₄⁺ or NO₃⁻. ¹⁵N was removed from the water at an almost identical rate and magnitude as ¹⁴N. Differences in accumulation of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ in the tissue reflected disappearance from the water; ¹⁵N from NH₄⁺ accumulated faster and reached an atom % twice that of ¹⁵N from NO₃⁻. This outcome suggested that when NH₄⁺ and NO₃⁻ were supplied in equal concentrations, more NH₄⁺ was taken up and assimilated. The ability to take up high concentrations of NH₄⁺, and NO₃⁻ simultaneously is important for bloom-forming species of estuarine macroalgae subject to multiple nutrient species from various sources.     

Potential difference responses due to K+, Na+ and Cl− changes in Bullfrog antrum with and without HCO3−

The effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution was studied in bullfrog antrum with and without HCO₃^? in nutrient. In 25 mM HCO₃^? (95% O₂/5% CO₂) and in zero HCO₃^? (100% O₂), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K⁺ or from 81 to 8.1 mM Cl^? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO₃^?. A decrease from 102 to 8 mM Na⁺ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO₃^?. In contrast, change from 4 to 40 mM K⁺ gave initial anomalous PD response and change from 102 to 8 mM Na⁺, initial normal PD response with either zero or 25 mM HCO₃^?. Both responses were associated with (Na⁺ + K⁺)-ATPase pump and were greater in zero than in 25 mM HCO₃^?. Initial PD increases in zero HCO₃^? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO₃^? modifies conductance pathways of nutrient membrane.     

One-carbon metabolism in methanogens: evidence for synthesis of a two-carbon cellular intermediate and unification of catabolism and anabolism in Methanosarcina barkeri

One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term ¹⁴CH₃OH or ¹⁴CO₂ incorporation studies during growth and by cell suspensions. ¹⁴CH₃OH and ¹⁴CO₂ were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H₂-CO₂-methanol. ¹⁴CH₃OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas ¹⁴CO₂ was selectively incorporated into the C₁ moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, ¹⁴CH₄ and [3-¹⁴C]alanine synthesized from ¹⁴CH₃OH during growth shared a common specific activity distinct from that of CO₂ or methanol. Cell suspensions synthesized acetate and alanine from ¹⁴CO₂. The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of ¹⁴CH₃OH or ¹⁴CO₂ fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from ¹⁴CH₃OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from ¹⁴CO (~1 nmol/min per mg of protein) and an isotopic exchange between CO₂ or CO and the C-1 of pyruvate. Acetate synthesis from ¹⁴CO was stimulated by methyl B₁₂ but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 μmol/min per mg of protein) and acetate kinase (>0.14 μmol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.     

Diurnal change of tartrate dissimilation during the ripening of grapes

l(+)-tartrate-[U-¹⁴C] or sucrose-[U-¹⁴C] was fed into grape berries and ¹⁴CO₂ evolution was determined. ¹⁴CO₂ evolution front l(+)-tartrate-[U-¹⁴C] was slightly higher in mature than immature berries, and that from sucrose-[U-¹⁴C] was higher in immature than mature ones. ¹⁴CO₂ evolution from l(+)-tartrate-[U-¹⁴C] was irregular throughout the day until 2 or 3 weeks after flowering. This stage shifted to regular ¹⁴CO₂ evolution until 6 or 7 weeks after flowering, and the mode of ¹⁴CO₂ evolution showed diurnal variation; higher in the day than at night. Then the stage without variation of ¹⁴CO₂ evolution followed 10 weeks after flowering. These observations indicate that tartrate is not biochemically inert in grape berries, while the amount of ¹⁴CO₂ evolution from sucrose-[U-¹⁴C] was higher at night than in the day through the whole ripening process, except in the early stage.     

Emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N₂ and N₂O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO₃^–, NO₂^– or NH₄⁺ -containing medium was investigated. ¹⁵N contents of gas sampled from gas-sealed pots, in which the plants were grown on ¹⁵N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N₂ was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO₃^–-containing medium. N₂O emission from clone 271 grown on NO₃^–-containing medium was detected, but not from the wild-type plants. The N₂O emission rate of clone 271 was 106 ng N₂O mg^–1 incorporated N week^–1 and the N₂O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N₂O was found from clone 271 or wild-type plants grown on medium containing NH₄⁺. Emission of N₂O also was detected from clone 271 grown on NO₂^–-containing medium and its emission rate increased with increasing NO₂^– levels in plants. We speculate that NO₃^– is reduced to NO₂^– and that a part of NO₂^– is metabolized to N₂O in clone 271.     

Estradiol -17-β enhances the formation of {3H}-PGF2α from {3H}-PGE2 in the uterus isolated from ovariectomized rats

Formation of {³H}-PGF_2α and {³H}-13,14,dihydro-15-keto-PGF_2α from {³H}-PGE₂ by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE₂ + NADPH + {³H}-PGE₂ + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus.Spayed animals were injected with progesterone (P) or with estradiol-17-β (E₀) at a dose of 1.0 or 50.0 ug. Conversion of {³H}-PGF_2α to {³H}-PGE₂ or to {³H}-13,14,dihydro-15-keto-PGF_2α did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 ug E₀. However, 50.0 ug E₀ induced a significant oversion after 30 (P < 0.01) and 60 (P < 0.001) min of incubation.It is concluded that E₀, at the 50.0 ug dose, but not the 1.0 ug dose of E₀, nor progesterone, stimulated conversion of {³H}-PGE₂ into {³H}-PGF_2α or {³H}-13, 14,dihydro-15-keto-PGF_2α, presumably through the activity of the enzyme PGE₂-9-keto-reductase.     

Charcoal-catalyzed transfer of tritium into 3H2O from regiospecifically-labeled 2-hydroxyestradiol in the presence of thiols

Charcoal was found to catalyze the release of ³H₂O from [1-³H]2-hydroxyestradiol-17β ([1-³H]2-OHE₂) or [4-³H]2-hydroxy-estradiol-17β ([4-³H]2-OHE₂) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of ³H₂O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of ³H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of ³H₂O from [4-³H]2-OHE₂ in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.     

Convergent pathways of gibberellin A1 biosynthesis in Brassica

[²H, ³H]Gibberellin A₄ (GA₄) or [²H, ³H] GA₉ were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [²H]GA₉ application, [²H]GA₅₁, [²H]GA₂₀ and [²H]GA₄ were identified as products by GC-MS, while [²H]GA₃₄ and [²H]GA₁ were formed from [²H]GA₄. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA₁ biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA₄ or GA₂₀, the latter of which can originate from GA₉ or from GA₁₉.     

Half molecules of serine-specific transfer ribonucleic acids from yeast

The preparation and analysis of half molecules from tRNA^Ser are described. Two pG-halves were isolated which differed only in the presence or absence of an acetyl group on the cytidylic acid residue at position 12. The CCA-half derived from tRNA₁^Ser was isolated pure, while the CCA-half derived from tRNA₂^Ser was isolated as a mixture with the CCA-half from tRNA₁^Ser from which the terminal CpCpA had been cleaved off.The acceptor activity of the combined complementary half molecules was 90% of the one of intact tRNA^Ser. The Michaelis constant and maximal velocity of amino-acylation were found to be identical for tRNA^Ser and the combined fragments.When half molecules were present at different ratios in aminoacylation studies it was found that one pG-half molecule can mediate the charging of several CCA-half molecules. There are indications that the CCA-half molecule alone can accept some serine. The CCA-half molecule alone can be aminoacylated to a rather high degree in the presence of an excess of tRNA_ox^Ser or tRNA^Ser-a and to a small degree in the presence of tRNA_ox^Ala (yeast) but not at all in the presence of tRNA_ox^Phe or tRNA_ox^Val (E. coli).Combinations of half molecules from tRNA^Ser with the opposite half molecules from tRNA^Phe could not be aminoacylated with Ser or Phe or 15 other amino acids although one of the combinations was well associated according to gel electrophoresis and differential melting curves.     

Presence of a Na+/H+ antiporter in Methanobacterium thermoautotrophicum and its role in Na+ dependent methanogenesis

Methane formation from H₂/CO₂ by methanogenic bacteria is dependent on Na⁺ ions. In this communication it is shown with Methanobacterium thermoautotrophicum that a Na⁺/H⁺ antiporter plays a role in methane formation from H₂ and CO₂ and in the regulation of the ΔpH. This is based on the following findings:

1. Li⁺ ions, an alternative substrate of Na⁺/H⁺ antiporters, could replace Na⁺ in stimulating methanogenesis from H₂ and CO₂.
2. Harmaline, amiloride, and NH ₄ ⁺ , which are inhibitors of Na⁺/H⁺ antiporters, inhibited methanogenesis; inhibition was competitive to Na⁺ or Li⁺.
3. Addition of Na⁺ or Li⁺ rather than of other cations to cell suspensions resulted in an acidification of the suspension medium. The rate and extent of acidification was affected by those inhibitors, which inhibited methanogenesis competitively to Na⁺ or Li.
4. During methane formation from H₂ and CO₂ the generation of a ΔpH (inside alkaline) was dependent on the presence of Na⁺ or Li⁺. However, methanogenesis was also dependent on Na⁺ or Li⁺ under conditions where ΔpH was zero.
5. ATP synthesis driven by an electrogenic potassium efflux was significantly enhanced in the presence of Na⁺ or Li⁺. Na⁺ or Li⁺ were shown to prevent acidification of the cytoplasm under these conditions.

     

P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

We aimed to study the role of the nucleotide receptor P2Y₂R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y₂^+/+ and P2Y₂^-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y₂^+/+ and P2Y₂^-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4⁺ T lymphocytes from P2Y₂^+/+ and P2Y₂^-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y₂^-/- mice as in P2Y₂^-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y₂^-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y₂^-/- and P2Y₂^+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y₂^-/- T cells proliferate less and secrete less cytokines than the P2Y₂^+/+ one. We further found that antigen-presenting cells of P2Y₂^-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y₂^-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.     

Superoxide dismutase: a comparison of rate constants

O₂^?was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂^? was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂^? by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂^? with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m^?1 sec^?1 at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂^? in the 10^?8m → 10^?13m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂^? concentrations in the 10^?5m range. The second order rate constant for the enzymatic dismutation of O₂^? is thus independent of the concentration of O₂^? in the range 10^?5 → 10^?13m.Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.     

The effect of different N substrates on biological N2O production from forest and agricultural light textured soils

N₂O emissions from two slightly alkaline sandy soils, from arable land and a woodland, were determined in a laboratory experiment in which the soils were incubated with different sources of nitrogen, with or without glucose, and with 0, 1 and 100 mL C₂H₂ L^-1. Large differences in the rate of N₂O production were observed between the two soils and between the different N treatments. The arable soil showed very low N₂O emissions derived from reduced forms of N as compared with the N₂O which was produced when the soil was provided with NO ₂ ^- or NO ₃ ^- and a C source, suggesting a very active denitrifier population. In contrast, the woodland soil showed a very low denitrification activity and a much higher N₂O production derived from the oxidation of NH ₄ ⁺ and reduction of NO ₂ ^- by some processes probably mediated by autotrophic or heterotrophic nitrifiers or dissimilatory NO ₂ ^- reducers. In both soils, the highest N₂O emissions were induced by NO ₂ ^- addition. Those emissions were demonstrated to have a biological origin, as no significant N₂O emissions were measured when the soil was autoclaved.     

Ammonia volatilization from cattle slurry following surface application to grassland

Three experiments were conducted using a system of small wind tunnels to measure ammonia (NH₃) volatilization from cattle slurry after surface application to land. In each experiment slurry was applied at a rate equivalent to 80 m³ ha^-1, providing the equivalent of approximately 100 kg NH₄ ⁺-N ha^-1. The first experiment compared NH₃ volatilization from the liquid fraction obtained by mechanical separation of slurry with that from unseparated slurry. The total NH₃ loss over six days from unseparated and separated slurry were very similar, being 38 and 35% respectively of the NH₄ ⁺-N applied. For the first five hours, the rate of NH₃ loss was higher from the unseparated slurry, thereafter it was consistently lower. In the second experiment, slurry was ponded in a tray to examine whether impeded infiltration or changes in the NH₄ ⁺ concentration or overall pH of the slurry influenced the rapid decline in rate soon after application that is characteristic of NH₃ volatilization from animal slurries applied to land. It appeared, however, that other factors such as resistance to diffusion within the slurry and/or at the slurry surface were mostly responsible for the rapid decline in rate. In the third experiment, in which NH₃ volatilization was measured from slurry applied to grassland or bare soil, the total loss from slurry applied to grassland was approximately 1.5 times that from slurry applied to bare soil.     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Effect of stress on sulfated glycol, metabolites of brain norepinephrine.

The present study examined the effect of footshock stress on the formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihydroxyphenylglycol (DOPEG-SO₄). Rats receiving intraventricular injections of either ³HNE or Na₂³⁵SO₄ prior to 0.5 hour of footshock showed significant and comparable increases in both sulfated glycols labeled with ³H or ³⁵SO₄. Elevations were greatest in the hypothalumus using Na₂³⁵SO₄. In pheniprazine pretreated rats footshock did not increase the production of MOPEG-³⁵SO₄ from intraventricular labeled sulfate given alone or in combination with various doses of exogenous MOPEG. The results indicate that neuronally released brain NE is metabolized to form both MOPEG-SO₄ and DOPEG-SO₄. The increase in these metabolites results from an increased glycol production and not from a stress-induced activation of brain sulfation mechanisms.     

Leaching of solutes by the action of acidic rain: a comparison of efflux from twigs and single needles of Picea abies (L. Karst.)

Abstract. Leaching of inorganic cations (K⁺, Mg²⁺) and in some cases of inorganic anions and sugars from detached twigs and single needles of spruce Picea abies L. Karst.) in the presence of acid rain (H₂SO₄, 1 mol m^?3) or salt solutions (Na₂ SO₄, 1 mol m^?3) was examined under laboratory conditions. Cation leaching (as percentage of the total water soluble ion content of the tissue per hour) was: K⁺: 0.01-0.02%; Mg²⁺: 0.005-0.01%; Ca²⁺: 0.1-0.2%. Leaching rates of anions were even lower than that and concentrations in the leachate were often below the detection limit of anion chromatography. Spraying with H₂SO₄ (pH 2.95, 1 mol m^?3) increased leaching only transiently. Similar effects were found when Na₂SO₄ was used instead of H₂SO₄. The transiently enhanced leaching was apparently due to H⁺/cation or cation/cation exchange at the twig or leaf surfaces. Feeding of K⁺ or Al³⁺ through the stems increased leaching of all cations within a few hours, again demonstrating rapid ion exchange in the apoplast. Leaching of potassium and magnesium from single needles occurred at similar relative rates as from twigs. Loss of Ca²⁺ ions, however, was even smaller from needles than from twigs. Apparently, a large part of the Ca²⁺ lost from twigs originated from the bark and not from the needles. Efflux of ions from longitudinal needle sections was about 1000 times taster than the rates obtained with intact needles, indicating that the cuticle was the main barrier Preventing solute loss. In relation to the total amount of mineral nutrients in trees, leaching is considered to be too small to be the primary cause of damage to trees stressed by acid rain, as has been suggested in the literature.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.     

Nitroxide Sod-Mimics: Modes of Action

Low molecular weight superoxide dismutase mimics have been shown to afford protection from oxidative damage. Such SOD-mimics can readily permeate cell membrane achieving sufficiently high levels both inside and outside the cell to effectively detoxify intracellular O^?₂. Preliminary findings also indicated that metal-based and metal-free SOD-mimics can protect hypoxic cells from H₂O₂-induced damage. The present study explored the possibility that SOD-mimics such as desferrioxamine-Mn(III) chelate [DF-Mn] or cyclic nitroxide stable free radicals could protect from O^?₂-independent damage. Killing of monolayered V79 Chinese hamster cells was induced by H₂O₂ or by t-butyl hydroperoxide (t-BHP) and assayed clonogenically. Neither catalase nor native SOD protected the cells from t-BHP. In contrast. both DF-Mn and cyclic nitroxides protected suggesting cytotoxic processes independent of O^?₂ or of O^?₂ -derived active species. The inhibition of the damage by both metal-free and metal-based SOD mimics is attributable to either SOD-mimic reacting with reduced transition metal to block the Fenton reaction and/or intercepting and detoxifying intracellular organic free radicals.