Stimulation Of O2 Uptake Research Articles

Peroxidase-like activity of uncoupled cytochrome P450: Studies with bilirubin and toxicological implications of uncoupling

The NADPH-dependent consumption of O2 by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O2 molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O2 uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O2 and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Abstract 440: Myoglobin in breast cancer: Regulation, function and relevance

Abstract Background Following an accidental observation, we aimed to analyse expression, regulation and function of myoglobin (Mb) in breast cancer. Methods Immunohistochemistry/Immunofluorescence, microdissection, transmission electron microscopy, Western blot, qPCR, cell culture (under normoxia and hypoxia), shRNA and siRNA knockdown, in silico data mining, high resolution respirometry. Functional assays: proliferation, migration and colony formation. Results Mb protein is de novo expressed in 71% of invasive breast carcinomas (n=917) in association with the hypoxia inducible factor 2α (HIF-2 α), carbonic anhydrase IX and a better prognosis. Expression of Mb in breast cancer was confirmed on mRNA level and occurred irrespective of rhabdomyoid differentiation and was inducible by prolonged hypoxia in breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7) with a median change fold of 3.4 after 72 hours. Interestingly, hypoxically induced MB mRNA originated from a novel, alternative transcription start site 6 kb upstream of the genuine ATG codon. Functionally, stable myoglobin knockdown in MDA-MB468 cells was associated with a stimulated O2 uptake during mild hypoxia, yet did not impact the O2 diffusion gradient in these small cells during limiting conditions. Knockdown of Mb also conferred a significant retardation of the cells’ in vitro motility to close an inflicted wound within the normoxic culture. Conclusions Regarding cancer cells, unconventional functions of myoglobin, not directly related to the transport of oxygen, thus need to be envisioned. These unprecedented findings may have fundamental implications for our understanding of non-muscle Mb in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 440.

Sulfur, carbon, and nitrogen cycling in faunated marine sediments impacted by repeated organic enrichment

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 400:37-53 (2010) - DOI: https://doi.org/10.3354/meps08400 Sulfur, carbon, and nitrogen cycling in faunated marine sediments impacted by repeated organic enrichment T. Valdemarsen*, E. Kristensen, M. Holmer Institute of Biology, University of Southern Denmark, 5230 Odense M, Denmark *Email: valdemarsen@biology.sdu.dk ABSTRACT: The effects of organic enrichment on sediment biogeochemistry were studied in sediment mesocosms with indigenous populations of infauna. Two types of sediments, differing in content of reactive Fe, were subjected to weekly pulses of labile organic matter (fish feed), corresponding to an average organic loading of ~350 C m–2 d–1 and ~50 mmol N m–2 d–1 for a period of 65 d. The aims of this experiment were 3-fold: (1) to study the metabolic capacity for organic C and N mineralization at the sediment–water interface; (2) to study the impact of organic enrichment on spatial and temporal sulfide accumulation; and (3) to estimate the importance of Fe-driven sulfide buffering for sulfide accumulation. Organic enrichment stimulated solute exchange between sediment and water within 10 d, resulting in 5 to 8 times stimulated O2 uptake and CO2 efflux, indicating a fast response time of surface-associated microbial consortia. Both 35S-SO42– radiotracer essays and sulfide microprofiles confirmed that added organic matter was primarily oxidized by sulfate reduction. However, despite high sulfate reduction of up to 70 mmol m–2 d–1, sulfide only accumulated to 1–2 mM in the upper 2 cm of enriched cores. In Fe-poor sediment, produced sulfide was reoxidized with O2 or NO3– at the sediment–water interface, whereas in Fe-rich sediment, sulfide was oxidized with O2, NO3–, or Fe oxides in a <2 mm thick suboxic zone or precipitated with dissolved Fe2+. By the end, up to 92 and 100% of added organic C and N, respectively, was mineralized, suggesting a high metabolic capacity for organic matter mineralization in faunated sediments. Furthermore, whereas sulfide precipitation was insignificant in low-Fe sediment, up to 63% of sulfide precipitated with Fe2+ in high-Fe sediment, suggesting a high capacity for sulfide mitigation in faunated Fe-rich sediment. KEY WORDS: Sulfide buffering · Benthic fluxes · Decomposition · Eutrophication · Fish feed · Sulfate reduction Full text in pdf format PreviousNextCite this article as: Valdemarsen T, Kristensen E, Holmer M (2010) Sulfur, carbon, and nitrogen cycling in faunated marine sediments impacted by repeated organic enrichment. Mar Ecol Prog Ser 400:37-53. https://doi.org/10.3354/meps08400 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 400. Online publication date: February 11, 2010 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2010 Inter-Research.

Chironomid larvae stimulate biogeochemical and microbial processes in a riverbed covered with fine sediment

We determined if the influence of chironomid larvae on biogeochemical and microbial processes in fine surface sediments of a riverbed depended on the characteristics of the sedimentary habitat. The influence of chironomids was measured on sediment reworking, biogeochemical processes and bacterial characteristics in infiltration sediment columns having three different surface sediments (with different organic matter characteristics). The results showed that chironomids exhibited similar bioturbation activities (construction and irrigation of U-shaped tubes in the upper layers of sediments) and significantly stimulated O2 uptake and percentage of active bacteria in all sediment treatments. In contrast, the release of NH4 + in anoxic sediment layers was strongly increased by chironomids in the presence of organicrich sediments, whereas they had no effect on this process with organic-poor sediments. Our results indicated that the influence of chironomids on biogeochemical processes in river bed sediments are linked to the redox conditions of the system. Moreover, when the effects of chironomid larvae are compared to those measured with tubificid worms in the same experimental conditions, we conclude that the mode of bioturbation (sediment reworking, biogenic structure building, burrowing depth, bioirrigation) can also determine the impact of benthic animals on microbial activities and biogeochemical processes in the sediment.

Effect of cobalt on growth, pigments and the photosynthetic electron transport in Monoraphidium minutum and Nitzchia perminuta

The unicellular green alga Monoraphidium minutum and the diatom Nitzschia perminuta were cultured under different concentrations of Co2+. Growth and pigment content were slightly increased at low concentrations and inhibited by high Co2+ concentrations. The results concerning the effect of different concentrations of Co2+ on photosynthetic O2 evolution showed a reduction in the amount of O2 evolved by each alga in response to increasing Co2+ concentrations. However, an increase in O2 evolution for both M. minutum and N. perminuta was observed at relatively low Co2+ concentrations. Photosynthetic electron transport in M. minutum was more sensitive to Co2+ toxicity than in N. perminuta. On the other hand, the effect of the heavy metal on respiration showed that higher Co2+ concentrations were inhibitory to O2 uptake by the two algal species. Low Co2+ concentrations stimulated O2 uptake by M. minutum throughout the experimental period. However, in N. perminuta, different concentrations of Co2+ led to a reduction of O2 uptake. To localize the action site of Co2+ in the photosynthetic electron transport chain, the fluorescence induction technique was carried out. According to the results obtained, the inhibitory action of Co2+ is located on the acceptor side of PSII for both M. minutum and N. perminuta.

Hypercholesterolemia and chronic anxiety disorder

This chapter discusses the oxystat technique in study of reactive oxygen species. The oxystat system allows samples to be collected from the filtrate as well as directly from the incubation chamber. Thus, the formation of products of oxidative reactions in biological material, such as malon- dialdehyde, can easily be determined during the course of incubation. Furthermore, by putting the incubation chamber directly in front of the photomultiplier of a single-photon counting apparatus, low-level chemiluminescence emitted by electronically excited species such as singlet molecular oxygen (1O2) and triplet carbonyls (3RO) can be detected. Finally, the stimulation of O2 uptake may serve as an additional indicator for the formation of reactive oxygen species. The oxystat system consists of an incubation chamber, an O2 sensor, a pump, and a computer. The water-jacketed incubation chamber is made of plexiglass. It is equipped with a magnetic stirrer and a port for the O2 sensor. At the top two stainless steel needles serve as entrances. The inlet for the O2-saturated infusion medium ends directly above the magnetic stirring bar. The second needle, which does not project into the chamber, is used as an injection port for the addition of reagents and for taking samples of the incubation mixture. The bottom of the incubation chamber is equipped with a membrane holder covered by a filter membrane. The actual PO2 in the incubation chamber is determined by a polarographic O2 sensor.

Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro.

Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks. Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF. Ascorbate catalyzed one-electron reduction to nitro anion radicals which reacted with molecular O2 to yield superoxide. Rates of O2 uptake with 9-oxo-2,4,7-triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those, respectively, with equivalent concentrations of nitrofurazone, a classical substrate. Superoxide formation was indicated by the approximately 75% regeneration of O2 upon addition of superoxide dismutase and catalase. 9-Oxo-2,4,7-triNF stimulated O2 uptake in the presence of XO/NADH with typical Michaelis-Menten kinetics with an apparent Km of 0.476 +/- 0.054 microM versus a Km of 6.18 +/- 0.719 microM for nitrofurazone. HPLC analyses of products from reduction catalyzed by XO or diaphorase of Clostridium with NADH showed the following trends for the rates of amine formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2-NF > 2-NF; 2,7-diNF > 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF. Little or no amine was formed in 95% O2, suggesting O2-labile intermediates. The data herein suggest that oxidation at C-9 and multiple nitro groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead to genotoxic effects.

Transfers of N metabolites across the ovine liver in response to short-term infusions of an amino acid mixture into the mesenteric vein

The effect of acute (4.5 h) infusions into the mesenteric vein of an amino acid (AA) mixture, which simulated the composition of rumen microbial protein, on net transfers of NH3, urea and total AA across the portal-drained viscera (PDV) and liver in the ovine has been examined. Four wether sheep were surgically prepared with vascular catheters across the PDV and liver (Lobley et al. 1995) and were offered a basal diet of 1000 g grass pellets/d (approximately 1.4 × energy maintenance). Each animal was infused at weekly intervals with one of four dilutions of the AA mixture. These dilutions provided 0.44, 0.88, 1.32 and 1.84mmol AA-N/min infused, the lowest of which approximately doubled the net absorption of AA-N from the basal diet. Animals were treated with heparin to allow continuous collection of blood by peristaltic pump for 2 h preceding, and between 0.5–2.5 and 2.5–4.5 h after, the start of the AA infusions. Blood flow in the hepatic artery increased (100 v. 208 g/min; P = 0.002) in response to AA infusion, while hepatic portal venous flow decreased (2090 v. 1854 g/min; P = 0.006). The AA infusion also stimulated O2 uptake by the PDV (P &lt; 0.001) and liver (P = 0.016). Absorption across the PDV and hepatic removal of NH3 were unchanged between basal and amino acid infusion conditions. Urea-N removal across the PDV was unaltered, but hepatic production increased (P &lt; 0.001) with level of AA infusion. During infusions, net appearance of AA across the PDV was below the theoretical level. This may have been due to inhibition of AA uptake from the small intestine, and/or increased removal by the digestive tract of AA from the systemic circulation associated with greater arterial concentrations. Hepatic extraction of AA increased with level of infusion, both for total AA and those included in the infusate. Total hepatic urea-N production tended towards a maximum (estimated as 2 μmol N/g liver wet weight per min). The AA removed by the liver and not used for ureagenesis remained similar (170 μmol AA-N/min) between basal and AA infusions. This was presumed available for anabolic purposes (mainly synthesis of export proteins). The proportion of net AA-N appearance (absorption plus infused) across the PDV removed by the liver declined from 0.71 to 0.53 between basal and AA infusions. In contrast to findings from cattle (Wray-Cahen et al. 1997), increased AA infusion did not alter the net removal of glutamine across the liver. This may reflect differences between the studies in NH3: AA-N absorbed. Further differences between the cattle study and the current findings may relate to the different physiological state (pregnancy v. growth), which may alter the partition of AA between anabolic and catabolic fates.

Transfers of N metabolites across the ovine liver in response to short-term infusions of an amino acid mixture into the mesenteric vein.

The effect of acute (4.5 h) infusions into the mesenteric vein of an amino acid (AA) mixture, which simulated the composition of rumen microbial protein, on net transfers of NH3, urea and total AA across the portal-drained viscera (PDV) and liver in the ovine has been examined. Four wether sheep were surgically prepared with vascular catheters across the PDV and liver (Lobley et al. 1995) and were offered a basal diet of 1000 g grass pellets/d (approximately 1.4 x energy maintenance). Each animal was infused at weekly intervals with one of four dilutions of the AA mixture. These dilutions provided 0.44, 0.88, 1.32 and 1.84 mmol AA-N/min infused, the lowest of which approximately doubled the net absorption of AA-N from the basal diet. Animals were treated with heparin to allow continuous collection of blood by peristaltic pump for 2 h preceding, and between 0.5-2.5 and 2.5-4.5 h after, the start of the AA infusions. Blood flow in the hepatic artery increased (100 v. 208 g/min; P = 0.002) in response to AA infusion, while hepatic portal venous flow decreased (2090 v. 1854 g/min; P = 0.006). The AA infusion also stimulated O2 uptake by the PDV (P < 0.001) and liver (P = 0.016). Absorption across the PDV and hepatic removal of NH3 were unchanged between basal and amino acid infusion conditions. Urea-N removal across the PDV was unaltered, but hepatic production increased (P < 0.001) with level of AA infusion. During infusions, net appearance of AA across the PDV was below the theoretical level. This may have been due to inhibition of AA uptake from the small intestine, and/or increased removal by the digestive tract of AA from the systemic circulation associated with greater arterial concentrations. Hepatic extraction of AA increased with level of infusion, both for total AA and those included in the infusate. Total hepatic urea-N production tended towards a maximum (estimated as 2 mumol N/g liver wet weight per min). The AA removed by the liver and not used for ureagenesis remained similar (170 mumol AA-N/min) between basal and AA infusions. This was presumed available for anabolic purposes (mainly synthesis of export proteins). The proportion of net AA-N appearance (absorption plus infused) across the PDV removed by the liver declined from 0.71 to 0.53 between basal and AA infusions. In contrast to findings from cattle (Wray-Cahen et al. 1997), increased AA infusion did not alter the net removal of glutamine across the liver. This may reflect differences between the studies in NH3: AA-N absorbed. Further differences between the cattle study and the current findings may relate to the different physiological state (pregnancy v. growth), which may alter the partition of AA between anabolic and catabolic fates.

Development of seed coat‐imposed dormancy during seed maturation in Cynoglossum officinale

The relationship between seed phenolics and appearance of seed coat–imposed dormancy during seed development in Cynoglossum officinale L. was studied. Up to 24 days after anthesis, seeds failed to germinate upon imbibition in Petri dishes at 25°C. At 44 days after anthesis, seeds were fully germinable; removal of seed coats did not improve their germination or O2 uptake. At 72 days after anthesis, mature seeds at the base of the cyme did not germinate unless their coats were removed. Removal of seed coat also stimulated O2 uptake at this harvest date. The methanol‐soluble phenolic content of the seeds increased during the early stages of seed development, in both the seed coat and the embryo. As seed development continued, the methanol‐soluble phenolic content of the embryo stabilized, but that of the seed coat declined. This decline was associated with an increase in the thioglycolic acid–soluble phenolics, presumably lignins, in the seed coat. These results suggest that polymerization of methanol–soluble phenolics into lignins in the seed coat during later stages of seed development renders the seed coat of C. officinale impermeable to 03, and thus keeps the seed dormant.

Stimulation of respiration inUlva lactuca by high concentrations of cadmium and zinc: Evidence for an alternative respiratory pathway

The effect of zinc on respiration of Ulva lactuca (previously incubated in filtered seawater, in the dark, for 18 h) was investigated using an oxygen electrode. When exposed to zinc (as sulphate) at concentrations up to 25 mM, zinc stimulated respiration with maximum stimulation occurring at 1.5 mM, and above 5 mM respiration was inhibited. When zinc was used in conjunction with the uncoupler DNP (dinitrophenol, 50 μM), the stimulatory effects were cumulative. Oligomycin (6 mM) reduced O2 uptake by ∼42% but the addition of zinc to oligomycin-treated alga restored respiration to near normal values, indicating that zinc did not achieve its uncoupling effect via the mitochondrial ATPase. When zinc was added to cyanide-treated U. lactuca, respiration was partially restored to normal rates. These results were similar to those obtained previously, which showed that cadmium (but not uncouplers such as DNP) increased respiration in cyanide-treated U. lactuca. Cadmium-stimulated respiration was abolished only when cyanide was used in conjunction with salicylhydroxamic acid (SHAM), which indicated the presence of an alternative oxidative, cyanide-resistant pathway that has not been reported previously for U. lactuca. It is suggested that Zn (or Cd) stimulated O2 uptake via the alternative oxidative pathway. © 1996 by John Wiley & Sons, Inc.

Thidiazuron stimulates germination and ethylene production inStriga hermonthica– comparison with the effects of GR-24, ethylene and 1-aminocyclopropane-1-carboxylic acid

AbstractSeed germination of the hemiparasitic angiospermStriga hermonthicais elicited by compounds present in the root exudates of the host plant. A variety of chemicals can substitute for the host-derived signal although the exact mechanism through which these act is unknown. In the present study, thidiazuron (TDZ), a cytokinin-active urea derivative was found to stimulate germination. This thidiazuron-induced germination was shown to be mediated by endogenous ethylene, by a similar mechanism to germination induced by host-root exudate and the synthetic stimulant GR-24. Comparing the effects of TDZ, host-root exudate, GR-24 and the ethylene precursor 1-aminocyclopropane-1-car-boxylic (ACC) suggests that ethylene production increases as a result of increased ACC synthesis and is consistent with a model forStrigaseed germination in which host-derived signals and other stimulants act by eliciting the synthesis of ethylene via a stimulation of ACC synthase activity. All germination stimulants tested trigger a rapid increase in O2uptake by conditioned seeds. The patterns of O2uptake following TDZ or GR-24 treatments show similarities and three distinct phases of respiration are apparent. In contrast, stimulation of O2uptake by ethylene results in a different pattern, with no distinct phases obvious following the initial burst of activity.

Increase in oxygen uptake due to arachidonic acid is oxygen dependent in the perfused liver

The purpose of this study was to determine whether the effect of arachidonic acid on hepatic O2 uptake is O2 dependent and which region of the liver lobule it affects. In livers perfused at normal flow rates, infusion of arachidonate increased O2 uptake significantly by about 20-25 mumol.g-1.h-1. When the flow rate was doubled to make the hepatic O2 gradient shallower, the increase in O2 uptake due to arachidonate was two to three times larger (i.e., approximately 50 mumol.g-1.h-1). In livers perfused in the retrograde direction, maximal rates of O2 uptake were about twofold higher in upstream pericentral than in downstream periportal regions, and arachidonic acid nearly doubled O2 uptake in downstream areas without affecting rates in upstream regions. Thus it is concluded that arachidonate stimulates O2 uptake in an O2-dependent manner. This effect was sensitive to an inhibitor of the lipoxygenase, nordihydroguaiaretic acid, in perfused liver but not in isolated hepatocytes. In addition, conditioned medium from Kupffer cells incubated at high O2 tension stimulated parenchymal cell O2 uptake. Furthermore, arachidonate increased intracellular Ca2+ in isolated Kupffer cells in a dose-dependent manner. These findings suggest that eicosanoids produced by nonparenchymal cells participate in a hepatic O2 sensor mechanism involving Ca2+ that regulates O2 uptake by parenchymal cells in the liver.

Hormones increase oxygen uptake in periportal and pericentral regions of the liver lobule.

The effect of several hormones known to alter intracellular free Ca2+ on rates of O2 uptake in periportal and pericentral regions of the liver lobule was studied in the perfused liver. Regional O2 uptake was measured by stopping the flow and monitoring the decrease in O2 concentration. When perfusion was in the anterograde direction, basal rates of O2 uptake were two to three times higher in periportal than in pericentral regions, and phosphorylase alpha activity, which increases as a function of intracellular free Ca2+ levels, was higher in periportal regions. In contrast, when perfusion was in the retrograde direction, rates of O2 uptake were two to three times greater in pericentral regions. Infusion of epinephrine (0.1 microM) or angiotensin II (5 nM) increased the rate of O2 uptake nearly exclusively in downstream areas of the lobule where O2 tension was low. When perfusions were in the anterograde direction, epinephrine increased phosphorylase alpha activity significantly only in pericentral regions. Stimulation of O2 uptake by epinephrine was blocked by the alpha-adrenergic receptor blocker phentolamine (1 microM) but not by the beta-receptor blocker propranolol. Thus hormones that increase intracellular calcium stimulate O2 uptake predominantly in regions of the liver lobule where O2 tension is lowest, supporting the hypothesis that oxygen tension regulates O2 uptake in the liver via mechanisms involving intracellular free Ca2+.

Superoxide generation during phagocytosis by Acanthamoeba castellanii: similarities to the respiratory burst of immune phagocytes

Starved cultures of the soil amoeba Acanthamoeba castellanii suspended in phosphate-buffered saline exhibit stimulated O2 uptake in response to phagocytosis of either heat-killed yeast or latex beads. This phagocytosis-dependent (cyanide-insensitive) oxidative activity was observed when cells were isolated from either cyanide-stimulated or -inhibited cultures, and was therefore independent of the relative activities of the alternative mitochondrial oxidases known to exist in this organism. The extra O2 consumed during phagocytosis was stoichiometrically converted into O- 2 as detected by the rate of superoxide dismutase-inhibitable cytochrome c reduction. Phagolysosomal membranes isolated after uptake of latex beads were enriched in a b-type cytochrome which was spectrally similar to that present in immune phagocytes. Thus, the biochemical events during phagocytosis by either A. castellanii or immune phagocytes appear similar, suggesting that the ‘respiratory burst’ enzyme(s) responsible for O- 2 generation in these two cell types is structurally related.

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O2 uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O2-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the "residual component", operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O2-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O2 reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O2 uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O2 uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O2 uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O2 uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O2-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the "residual component" and the peroxidase-mediated O2 uptake. The peroxidase-mediated O2 uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the "residual component" 24%.

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.

Adenylate control of respiration in plants: the contribution of rotenone‐insensitive electron transport to ADP‐limited oxygen consumption by soybean mitochondria

The aim was to test the hypothesis that rotenone‐insensive electron transport (bypass of complex I) may underlie rapid state 4 (ADP‐limited) mitochondrial respiration. A comparison of mitochondria from soybean (Glycine max L. cv. Bragg) cotyledons and nodules showed that ADP‐sufficient (state 3) malate plus pyruvate oxidation by mitochondria from 7‐day‐old cotyledons was inhibited 50% by rotenone and state 4 rates were rapid, whereas nodule mitochondria were 80% inhibited by rotenone and had slower state 4 rates of malate plus pyruvate oxidation. Respiration of malate alone (pH 7.6) by cotyledon mitochondria was slow, especially in the absence of ADP; subsequent addition of pyruvate dramatically increased state 4 oxygen uptake concomitant with a rapid rise in mitochondrial NADH (determined by fluorimetry). Rotenone had no effect on this increased rate of state 4 respiration. The rate of malate oxidation by nodule mitochondria was relatively rapid compared with cotyledon mitochondria. The addition of pyruvate in state 4 caused a slow increase in matrix NADH and only a slight stimulation of oxygen uptake. Rotenone inhibited state 4 malate plus pyruvate oxidation by 50% in these mitochondria. From a large number of cotyledon and nodule mitochondrial preparations, a close correlation was found between the rate of state 4 oxygen uptake and rotenone‐resistance. During cotyledon development increased rotenone‐resistance was associated with an increase in the alternative oxidase. Addition of pyruvate to cotyledon mitochondria, during state 4 oxidation of malate in the presence of antimycin A, significantly stimulated O2 uptake and also almost eliminated respiratory control. Such combined operation of the rotenone‐insensitive bypass and the alternative oxidase in vivo will significantly affect the extent to which adenylates control the rate of electron transport.

Stimulation of oxygen uptake by glucagon is oxygen dependent in perfused rat liver.

Livers from well-fed female Sprague-Dawley rats (100-150 g) were perfused at flow rates of 4 or 8 ml.g liver-1.min-1 to deliver O2 to the organ at various rates. During perfusion at normal flow rates (4 ml.g-1.min-1), glucagon (10 nM) increased O2 uptake in perfused liver by approximately 40 mumol.g-1.h-1. In contrast, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 when livers were perfused at high flow rates. Increase in O2 uptake was directly proportional to flow rate and was blocked partially by infusion of phorbol myristate acetate (100 nM) before glucagon. Increase in O2 uptake due to elevated flow was not due to enhanced glucagon delivery, since infusion of 120 nM glucagon at normal flow rates only increased O2 uptake by approximately 40 mumol.g-1.h-1. On the other hand, when O2 tension in the perfusate was manipulated at normal flow rates, the stimulation of O2 uptake by glucagon increased proportional to the average O2 tension in the liver. Infusion of 8-bromo-adenosine 3',5'-cyclic monophosphate (BrcAMP; 25 microM) also increased O2 uptake more than twice as much at high compared with normal flow rates. In the presence of angiotensin II (5 nM), a hormone that increases intracellular calcium, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 at normal flow rates. Infusion of glucagon or BrcAMP into livers perfused at normal flow rates increased state 3 rates of O2 uptake of subsequently isolated mitochondria significantly by approximately 25%. In contrast, perfusion with glucagon or BrcAMP at high flow rates increased mitochondrial respiration by 50-60%. Glucagon addition acutely to suspensions of mitochondria, however, had no effect on O2 uptake. These data are consistent with reports that glucagon administration in vivo or treatment of intact cells with glucagon increases O2 uptake of subsequently isolated mitochondria, a phenomenon that can account for the observed increase in O2 uptake in livers perfused at high flow rates with glucagon. Furthermore, these results are consistent with the hypothesis that the effect of glucagon on mitochondria is O2 dependent in the perfused liver. This is most likely due to an effect of intracellular calcium on a mechanism mediated via cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation between cytosolic free calcium and respiratory rates in cardiac myocytes

Rates of O2 uptake of isolated rat cardiac myocytes were determined as a function of cytosolic free Ca2+ concentration ([Ca2+]c) that was estimated from intracellular quin2 fluorescence. [Ca2+]c was increased by depolarization with K+ or veratridine. In each case, there was a correlation between increase in [Ca2+]c and stimulation of O2 uptake. Apparent exception seen on raising K+ were resolved on the of an effect of osmolality on O2 uptake rates. Increase in O2 uptake and [Ca2+]c by veratridine was sensitive to variation of extracellular Na+, Ca2+, and pH in a way that suggests a major involvement of the Na+-Ca2+ exchange: partial inhibition by 2.7 microM verapamil and total inhibition by 30 microM 3',4'-dichlorobenzamil were consistent with this conclusion. Attempts were made to assess the quantitative significance of direct activation of respiration by Ca2+ at the level of mitochondrial dehydrogenases vs. an indirect mechanism involving increased ADP generation. Ruthenium red, which blocks the former process but not the latter, gave a small decrease in O2 uptake rates. However, activation of oxidative phosphorylation by ADP was predominant under these conditions of profound and sustained depolarization, based on a lowered mitochondrial NADH content in response to veratridine.