Stimulation Of O2 Consumption Research Articles

Stoichiometry of regenerated nutrients differs between native and invasive freshwater mussels with implications for algal growth

Abstract Filter‐feeding mussels couple benthic and pelagic environments and create biogeochemical hot spots. Mussels may exert either top‐down control (via filtration) or bottom‐up stimulation (via biodeposition and excretion) of primary producers. Mussel metabolism may be species‐specific and the disappearance of native species or their replacement by invasive species may affect ecosystem functioning, notably gas exchange, nutrient fluxes, and stoichiometry at the sediment–water interface. In this study, we tested experimentally how native (unionids) and invasive (dreissenids) mussels, singly and in association, affect benthic fluxes of dissolved gas and nutrients in the light and in the dark, and indirectly phytoplankton growth from a freshwater estuary, the Curonian Lagoon, where the two species of mussel coexist. We show that native and invasive mussels stimulated O2 consumption and the production of total dissolved inorganic carbon and N2 increasing sediment heterotrophy and nutrient regeneration, with species‐specific effects on nutrient stoichiometry and algal growth. Ammonium and SiO2 exchanges were similar for both species, while soluble reactive phosphorus fluxes and excretion rates were significantly higher in sediments with dreissenids. Phytoplankton growth was also significantly higher in the presence of dreissenids as compared to unionids. Dreissenid mussels decreased the dissolved inorganic N to P ratio of regenerated nutrients and may favour the growth of cyanobacteria. Hence, the replacement of native with invasive mussels may produce large changes in benthic nutrient cycling and in phytoplankton growth and community composition.

Breaking the enzymatic latch: impacts of reducing conditions on hydrolytic enzyme activity in tropical forest soils

The enzymatic latch hypothesis proposes that oxygen (O2) limitation promotes wetland carbon (C) storage by indirectly decreasing the activities of hydrolytic enzymes that decompose organic matter. Humid tropical forest soils are often characterized by low and fluctuating redox conditions and harbor a large pool of organic matter, yet they also have the fastest decomposition rates globally. We tested the enzymatic latch hypothesis across a soil O2 gradient in the Luquillo Experimental Forest, Puerto Rico, USA. Enzyme activities expressed on a soil mass basis did not systematically decline across a landscape O2 gradient, nor did phenolics accumulate, the proposed mechanism of the enzymatic latch. Normalizing enzyme activities by C concentrations did suggest a decline in several enzymes as mean soil O2 decreased. However, relationships between hydrolytic enzymes and reducing conditions were scale‐dependent: enzymes displayed neutral to strongly positive relationships with reducing conditions and phenolics when comparing samples within sites, and enzyme activities in 18‐d anaerobic incubations generally exceeded those in aerobic soils despite a fourfold increase in phenolics. In summary, although O2 availability and the activities of some enzymes appeared to be related at landscape scales after accounting for differences in organic matter, reducing conditions and phenolic compounds did not appear to constrain soil hydrolytic enzyme activity at the scale of soil microsites, challenging the enzymatic latch hypothesis. Hydrolytic enzymes can be resilient to periodic anaerobiosis and may actually stimulate O2 consumption at the microsite scale. We suggest a critical re‐examination of mechanisms and the scale dependence of couplings between O2 and decomposition in terrestrial soils.

Effects of temperature and irradiance on a benthic microalgal community: A combined two‐dimensional oxygen and fluorescence imaging approach

The effects of temperature and light on both oxygen (O2) production and gross photosynthesis were resolved in a benthic microalgal community by combining two‐dimensional (2D) imaging of O2 and variable chlorophyll a (Chl a) fluorescence. Images revealed a photosynthetically active community with spatial heterogeneity at the millimeter scale. Irradiance strongly increased pore‐water O2 concentration, sediment net O2 production, and gross photosynthesis. The latter was derived from measurements of the electron transfer rate (rETR) in Photosystem II. The onset of light saturation for gross photosynthesis was approximately twofold higher than for net O2 production, reflecting significant light‐stimulated O2 consumption at higher light (> 75 µmol photons m−2 s−1). Temperature stimulated O2 consumption more than photosynthesis, turning the community more heterotrophic at elevated temperatures. Thus, the compensation irradiance (i.e., the irradiance at which community O2 production and consumption balance) increased fivefold (from 6 to 30 µmol photons m−2 s−1) with a temperature increase from 10°C to 25°C, corresponding to a temperature coefficient (Q10) of 2.9. Whereas net O2 production had a temperature optimum at ∼ 20°C, no optimum was observed for gross photosynthesis within the investigated range (10°C to 25°C). The resolved 2D net O2 production and rETR exhibited a significant exponential relationship, demonstrating predictable correlations between the net community production and gross photosynthesis for a complex microbial community, at different temperatures. The present imaging approach demonstrates a great potential to study consequences of environmental effects on photosynthetic activity and O2 turnover in complex phototrophic benthic communities.

Culture conditions and detachment of the fruit influence the effect of ethylene on the climacteric respiration of melon

The respiratory rise associated with ripening has been considered a defining feature of climacteric fruit. However, recent work has suggested that the respiration rate remains constant in melon fruit ripening whilst attached to the plant. To clarify the relationship between attachment and the respiratory climacteric, ethylene production and O2 consumption were monitored during the ripening of melon fruits grown in France and Australia. All fruit allowed to ripen after being detached from the vine produced both respiratory and ethylene climacterics. However, although ethylene production increased in attached fruit, the concurrent rise in respiration was either eliminated or reduced in magnitude. A positive relationship was found between ethylene levels and respiration rate. However, for a given rate of ethylene production the stimulation of respiration was less in attached than detached fruit. The application of exogenous ethylene to melons antisensed for ACC oxidase stimulated O2 consumption only if they were detached from the vine. These data also demonstrate that attachment to the plant inhibits the effects of ethylene on respiration. In addition, it was noticed that transgenic plants were better able to tolerate infection by powdery mildew than wild-type plants. As ethylene production was greater in attached fruit, it is further suggested that feedback inhibition of ethylene synthesis is reduced while fruit remain attached to the vine. It is concluded that the presence or absence of a respiratory climacteric in fruit attached to the vine is affected by environmental factors influencing plant development. These effects may be related to changes in gas permeance, turgor, or the supply of ‘plant factors’ translocated through the phloem. The results support the hypothesis that the respiratory climacteric is not an essential part of ripening, but an artefact caused by stress or detachment.

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON‐LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5‐fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron‐limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron‐limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O2 consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O2 consumption could be decreased by half by the addition of catalase, indicating that H2O2 is the product of the O2 consumption and that the increased O2 consumption is nonrespiratory. The stimulation of O2 consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe2+. Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O2 consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe2+, which is the product of ferric reductase activity. At saturating concentrations of Fe3+ chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe2+ and superoxide generation. Therefore, O2 consumption is not an integral part of the iron assimilation process.

Oxygen consumption oscillates in single clonal pancreatic beta-cells (HIT).

Based on population studies, we have hypothesized that changes in metabolism in pancreatic beta-cells precede changes in Ca2+. It is well known from single-cell Ca2+ studies that variable oscillatory patterns in Ca2+ occur in response to glucose stimulation. The present studies, using the clonal beta-cell line HIT-T-15, were undertaken to evaluate the relationship between glucose concentration, insulin secretion, and O2 consumption and to determine the Ca2+ dependency of glucose-induced changes in O2 consumption. In population studies, an excellent correlation was found between respiration and insulin secretion, with half-maximal values at approximately 1 mmol/l glucose for both respiration and secretion. In the absence of Ca2+, glucose stimulated O2 consumption but not insulin secretion. In single clonal beta-cells, a self-referencing O2 electrode was used to assess O2 consumption. Large-amplitude oscillations were found to occur in response to stimulation by glucose and were blocked by uncoupling respiration with carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). They were also blocked and respiration totally inhibited by antimycin A, an inhibitor of complex III of the respiratory chain. Half of the cells sampled (approximately 100 total) exhibited increased oscillatory O2 consumption in response to glucose. Oscillations in O2 occurred in response to glucose even in the absence of Ca2+, and their amplitude increased further on restoration of a normal extracellular Ca2+ level. These studies indicated that oscillatory O2 consumption was not dependent on Ca2+ but that the amplitude of the O2 oscillations increased in the presence of Ca2+, possibly reflecting the additional work involved in insulin secretion and Ca2+ pumping. These studies demonstrated, for the first time, a direct correlation between O2 consumption and insulin secretion, the oscillatory nature of O2 consumption in single cells, and the feasibility of using a highly sensitive noninvasive on-line self-referencing O2 electrode to monitor single beta-cell respiration.

Butane metabolism by butane-grown 'Pseudomonas butanovora'.

The pathway of butane metabolism by butane-grown 'Pseudomonas butanovora' was determined to be butane --> 1-butanol --> butyraldehyde butyrate. Butane was initially oxidized at the terminal carbon to produce 1-butanol. Up to 90% of the butane consumed was accounted for as 1-butanol when cells were incubated in the presence of 5 mM 1-propanol (to block subsequent metabolism of 1-butanol). No production of the subterminal oxidation product, 2-butanol, was detected, even in the presence of 5 mM 2-pentanol (an effective inhibitor of 2-butanol consumption). Ethane, propane and pentane, but not methane, were also oxidized. Butane-grown cells consumed 1-butanol and other terminal alcohols. Secondary alcohols, including 2-butanol, were oxidized to the corresponding ketones. Butyraldehyde was further oxidized to butyrate as demonstrated by blocking butyrate metabolism with 1 mM sodium valerate. Butyrate also accumulated from butane when cells were incubated with 1 mM sodium valerate. The pathway intermediates (butane, 1-butanol, butyraldehyde and butyrate) and 2-butanol stimulated O2 consumption by butane-grown cells. 1-Butanol, butyraldehyde and butyrate supported growth of 'P. butanovora', as did 2-butanol and lactate.

Oxidative burst and electrolyte leakage induced by sulfhydryl blockers and by membrane permeabilizing reagents in different organs ofEgeria densa

Sulfhydryl blockers, such as N-ethylmaleimide, iodoacetate and heavy metals induce a transitory stimulation of O2 consumption and H2O2 production (oxidative burst) and a rapid release of electrolytes in leaves of various aquatic plants. The correlation between these two responses to N-ethylmaleimide or to Ag+ in separate organs and stages of leaf development was investigated inEgeria densa. Only adult leaves were able to respond to the sulfhydryl blockers with an oxidative burst, whereas this response was absent in immature growing leaves and in stem and root segments. In N-ethyl-maleimide- as well as in Ag+-treated adult leaves the oxidative burst was constantly associated with a relevant electrolyte leakage. These data are consistent with a model in which the SH reagent would first interact with a plasmalemma protein, leading to an increase in passive permeability to ions and to the activation of an oxidative enzyme of the type of the superoxide synthase described for granulocytes. In its turn, active-oxygen species produced by the activated oxidase might further damage the plasma membrane, increasing its passive permeability. Digitonin and nystatin, two reagents known to cause a permeabilization of lipid membranes, induced in adultE. densa leaves a transient increase in the rate of O2 consumption and H2O2 production and an electrolyte leakage very similar to those induced by sulfhydryl blockers. These effects, however, were not influenced by the flavin analogues diphenylene iodonium and quinacrine, and were partially inhibited by the presence of CN− and salicylhydroxamic acid, thus suggesting the involvement of a different oxidase in the oxidative burst elicited by these reagents.

Leishmania donovani attachment stimulates PKC-mediated oxidative events in bone marrow-derived macrophages.

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O2- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O2- and NO production and stimulation of O2 consumption. Optimal NO and O2- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O2- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment Activation of O2- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.

Interactions between respiration and inorganic phosphate uptake in phosphate‐limited cells of Chlamydomonas reinhardtii

Phosphate addition to P‐limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O2 consumption. The respiration rate continued to increase for several minutes after the addition of P1. Similar patterns of P1 stimulation of respiratory O2 consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O2 consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP‐dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P1, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P1, whereas exhaustion of extracellular P1 resulted in a rapid decline in the O2 consumption rate. These results are consistent with control of respiration in P‐limited cells occurring largely at the level of glycolysis.

Kupffer cell prostaglandin E2 stimulates parenchymal cell O2 consumption: alcohol and cell-cell communication.

Several studies have demonstrated that ethanol can increase hepatic O2 uptake (e.g., produce a hypermetabolic state); however, a complete explanation of this important phenomenon remains unclear. Here, the effect of conditioned media from Kupffer cells isolated from rats chronically exposed to ethanol on O2 consumption of normal parenchymal cells was studied to evaluate the possibility that cell-cell communication participates in the mechanism of the hepatic hypermetabolic state. Kupffer cells were isolated from rats fed either a liquid control diet or a diet containing ethanol. Kupffer cells were cultured for 4 h, and conditioned media were incubated with parenchymal cells isolated from untreated rats in a closed chamber with an O2 electrode. O2 consumption of parenchymal cells incubated in fresh media or conditioned media from Kupffer cells from untreated rats was approximately 30 microliters.h-1. 10(6) cells-1; however, values were increased by > 30% by conditioned media from Kupffer cells isolated from rats treated with ethanol. Indomethacin, nisoldipine, and boiling the conditioned media blocked this stimulation, suggesting the involvement of eicosanoids. Indeed, prostaglandin E2 (PGE2) added directly to parenchymal cells increased O2 consumption in a dose-dependent manner by nearly 60%. Furthermore, PGE2 levels in conditioned media from Kupffer cells isolated from ethanol-treated rats were elevated about twofold. The addition of endotoxin to cultured cells caused a similar phenomenon. Taken together, these data support the hypothesis that Kupffer cells are activated by ethanol treatment to release mediators such a PGE2, which stimulate O2 consumption in parenchymal cells, possibly by mechanisms involving bacterial endotoxin.

EXTRACELLULAR PEROXIDASE-MEDIATED OXYGEN CONSUMPTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The unicellular green alga Chlamydomonas reinhardtii Dang. displays a high capacity for salicylhydroxamic acid (SHAM)—stimulated O2 consumption, mediated by extracellular peroxidaie. Addition of exogenous NADH also resulted in stimulation of O2 consumption. The SHAM-and NADH-stimulated peroxidase activity was partially sensitive to inhibition by exogenous superoxide dismutase, ascorbate, and gentisic acid. These compounds did not inhibit O2 consumption in the absence of effectors. SHAM-and NADH-stimulated peroxidase activity also was sensitive to inhibition by cyanide, and cyanide titration curves indicated that O2 consumption by peroxidase was more cyanide-sensitive than O2 consumption by cytochrome oxidase. The differential sensitivity to cyanide was used to estimate partitioning of O2 consumption between mitochondrial respiration and extracellular peroxidase. We suggest that, despite a large capacity for peroxidase-me-diated O2 consumption, peroxidase did not consume O2 at detectable rates in the absence of effectors. Therefore, in the absence of effectors, measured rates of O2 consumption represented the rate of mitochondrial respiration.

Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine.

Tri-iodothyronine (T3) and thyroxine (T4) as well as 3,5-di-iodothyronine (T2) stimulated O2 consumption by isolated perfused livers from hypothyroid rats at a concentration as low as 1 pM by about 30% within 90 min. Application of T2 resulted in a faster stimulation than with application of T3 or T4. Inhibition of iodothyronine monodeiodinase by propylthiouracil, thereby blocking the degradation of T4 to T3 and of T3 to T2, demonstrated that only T2 is the active hormone for the rapid stimulation of hepatic O2 consumption: T3 and T4 lost all of their stimulative activity, whereas T2 was as potent as in the absence of propylthiouracil. Perfusion experiments with thyroid-hormone analogues confirmed the specificity of the T2 effect. The nucleus is unlikely to contribute to the rapid T2 effect, as can be deduced from perfusion experiments with cycloheximide and lack of induction of malic enzyme by T2. In conclusion, a new scheme of regulation of mitochondrial activity is proposed: T2 acts rapidly and directly via a mitochondrial pathway, whereas T3 exerts its long-term action indirectly by induction of specific enzymes.

Regulation of immune-aggregate-stimulated hepatic glycogenolysis and vasoconstriction by vicinal dithiols

Evidence suggesting that vicinal dithiols regulate immune-aggregate-induced vasoconstriction and glycogenolysis in the perfused rat liver was obtained. Phenylarsine oxide (PhAsO) and other tervalent organic arsenicals inhibited in a dose-dependent manner hepatic glycogenolysis, vasoconstriction, Ca2+ mobilization and the stimulated O2 consumption caused by immune-aggregate infusion. Polar tervalent and quinquivalent arsenicals were less effective than hydrophobic arsenicals. Prior infusion of Fc- but not Fab-fragments of IgG prevented partially immune-aggregate-stimulated hepatic metabolism, suggesting that immune aggregates elicit hepatic metabolic responses through Fc gamma receptors. The inhibitory action of PhAsO on immune-aggregate-stimulated hepatic glycogenolysis was unique; inhibition of glycogenolysis was not observed when phenylephrine, isoprenaline or glucagon was used as a stimulant. Although PhAsO might be expected to sequester cellular thiols, no significant change in the oxidation-reduction state of the major cellular thiol, glutathione, was found during PhAsO infusion. In addition, PhAsO exerted its effects without producing changes in hepatic adenine nucleotides and cyclic AMP. Evidence suggesting the involvement of vicinal dithiols was obtained through thiol-competition experiments using mono- and di-thiols. PhAsO inhibition of IgG-aggregate-stimulated hepatic vasoconstriction and glycogenolysis was reversed significantly by infusion of 2,3-dimercaptopropan-1-ol at 3-fold molar excess, whereas 2-mercaptoethanol at 40-fold molar excess was ineffective. The results of the present study provide evidence documenting the participation of vicinal dithiols during the coupling of hepatic immune-aggregate clearance by Kupffer cells with vasoconstriction of the hepatic vasculature (e.g. endothelial cells) and glycogenolysis (e.g. parenchymal cells).

Redox activity and peroxidase activity associated with the plasma membrane of guard-cell protoplasts

Redox systems have been reported in the plasma membrane of numerous cell types and in cells from various species of higher plant. A search for a redox system in the plasma membrane of guard cells was therefore made in efforts to explain how blue light stimulates stomatal opening, a process which is coupled to guard cell H(+) efflux and K(+) uptake. The rates of O2 uptake by intact guard-cell protoplasts (GCP) of Commelina communis L., in the dark, were monitored in the presence of NAD(P)H since the stimulation of O2 consumption by reduced pyridine nucleotides is used as an indicator of the presence of a redox system in the plasma membrane. Oxygen consumption by intact GCP increased two- to threefold in the presence of NAD(P)H. The NAD(P)H-stimulation of O2 uptake was dependent on Mn(2+) and was stimulated 10- to 15-fold by salicylhydroxamic acid (SHAM). Catalase, cyanide and ascorbate, a superoxide scavenger, all individually inhibited the SHAM-stimulated O2 uptake. These are all characteristics of peroxidase activity although some of these features have been used to imply the presence of a redox system located in the plasma membrane. High levels of peroxidase activity (using guaiacol as a substrate) were also detected in the GCP and in the supernatant. The activity in the supernatant increased with time indicating that peroxidase was being excreted by the protoplasts. The properties of O2 uptake by the incubation medium after separation from the protoplasts were similar to those of the protoplast suspension. It is concluded that our observations can be more readily explained by peroxidase activity associated with the plasma membrane and secreted by the GCP than by the presence of a redox system in the plasma membrane of the protoplasts.

Zinc inhibition of respiratory burst in zymosan-stimulated neutrophils: a possible membrane action of zinc.

The effect of Zn2+ on the respiratory burst of rat pleural neutrophils was studiesd. Serum treated zymosan (STZ)-stimulated O2 consumption was inhibited by Zn2+ depending on hte Zn2+ concentration and time of cell incubation. Addition of Zn2+ after the stimulation of cells with STZ did not inhibit the O2 consumption Zn2+ failed to affect the cell viability or the opsonizing process of STZ, indicating that the inhibition of O2 consumption was not due to te direct cytotoxic action of Zn2+ or to the interaction of Zn2+ with STZ. In addition, the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzyme responsile for the respiratory burst of neutrophils, was not inhibited by Zn2+. These results suggested that Zn2+ may inhibit some process of the activation of NADPH oxidase.Equimolar Zn2+ 8-hydroxyquinoline complex (Zn-8HQ), which is known to be unable to penetrate the cell membrane, inhibited the O2 consumption more drastically than did Zn2+ alone. 8-Hydroxyquinoline alone did not cause significant inhibition of O2 consumption, indicating that the inhibitory effect of Zn2+ is potentiated by complexing with 8-hydrozyquinoline. The inhibition of O2 consumption Zn2+ was almost completely restored by washing and reincubating the Zn2+-treated cells in Zn2+-free medium. On the other hand, in the cells treated with Zn-8HQ, the same treatment of cell washing and reincubation only partially restored the O2 consumption. These results suggested that Zn2+ may be acting on the cell membrane of neutrophils.

Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro

Ascorbic acid may not only be involved as an antioxidant in the aqueous humour and lens but under certain conditions may also undergo 'pro-oxidant conversion' leading to the generation of oxidants. It is demonstrated that the bovine aqueous humour contains electron spin resonance (ESR) detectable levels of ascorbyl semiquinone free radical associated with the transition metal-catalysed oxidation of ascorbate. Cu2+ was found to be a more efficient catalyst of ascorbate oxidation than Fe2+ and was similarly more potent in inhibiting 86Rb uptake of lens epithelial cells in the presence of ascorbate. The metal-chelating agent diethylenetriaminepentaacetic acid (DETAPAC) inhibited metal-catalysed ascorbate oxidation and lens epithelial cytotoxicity. Riboflavin in the presence of light stimulates the formation of ESR-detectable ascorbyl semiquinone radicals and stimulates O2 consumption (oxidation) by ascorbate in a manner dependent upon the concentration of riboflavin. It is proposed that photoexcited riboflavin can perform a one-electron oxidation of ascorbate generating a riboflavin radical. This radical may then autoxidize generating superoxide anion. The riboflavin-mediated photoimpairment of 86Rb uptake of bovine lens epithelial cells was found to be dependent upon the presence of ascorbate. This 'pro-oxidant' activation of ascorbate by riboflavin is discussed in the context of previous studies on riboflavin phototoxicology.

Eosinophils Do Respond to fMLP

Eosinophils were isolated from normal human blood by separation over Percoll gradients, which resulted in eosinophil suspensions of a purity higher than 95% and recoveries of about 65%. Normal human eosinophils were found to respond to formyl-methionyl-leucyl-phenylalanine (fMLP) at concentrations greater than 10(-7) mol/L with an increase in the concentration of intracellular free calcium, oxygen consumption, nitroblue tetrazolium reduction, and chemiluminescence. The maximal response of eosinophils to fMLP was lower than that of neutrophils isolated from the same blood samples and required at least ten times as much fMLP as was needed for neutrophils. Low fMLP concentrations (approximately 10(-8) mol/L), which in themselves did not stimulate O2 consumption by either eosinophils or neutrophils, primed these cells to respond to a suboptimal concentration of another stimulus. Purification of eosinophils after treatment of whole blood with fMLP showed that these eosinophils had lost their ability to respond to fMLP. We conclude that normal eosinophils do respond to fMLP and that therefore fMLP should not be used to isolate eosinophils.

Eosinophils do respond to fMLP

Eosinophils were isolated from normal human blood by separation over Percoll gradients, which resulted in eosinophil suspensions of a purity higher than 95% and recoveries of about 65%. Normal human eosinophils were found to respond to formyl-methionyl-leucyl-phenylalanine (fMLP) at concentrations greater than 10(-7) mol/L with an increase in the concentration of intracellular free calcium, oxygen consumption, nitroblue tetrazolium reduction, and chemiluminescence. The maximal response of eosinophils to fMLP was lower than that of neutrophils isolated from the same blood samples and required at least ten times as much fMLP as was needed for neutrophils. Low fMLP concentrations (approximately 10(-8) mol/L), which in themselves did not stimulate O2 consumption by either eosinophils or neutrophils, primed these cells to respond to a suboptimal concentration of another stimulus. Purification of eosinophils after treatment of whole blood with fMLP showed that these eosinophils had lost their ability to respond to fMLP. We conclude that normal eosinophils do respond to fMLP and that therefore fMLP should not be used to isolate eosinophils.

Role of brown adipose tissue in thermogenesis induced by overfeeding a diet containing medium chain triglyceride.

The role of brown adipose tissue in the mechanism of medium chain triglyceride (MCT)-induced thermogenesis was investigated. Under anesthesia, the interscapular brown adipose tissue (IBAT) was excised in male Sprague-Dawley rats, and the animals were fitted with gastrostomy tubes. After a 10-day recovery period, the animals were divided into two groups: one group received a diet containing MCT as 50% of calories, and the other group received an isocaloric diet containing long chain triglyceride (LCT). The diets were fed for 6 wk at a level of calorie intake that was 150% of the ad libitum intake of a parallel control group. During the last week of the study, resting and norepinephrine (NE)-stimulated O2 consumption and CO2 production were measured in a Noyons diaferometer. At the end of 6 wk, the animals were weighed and killed. The individual fat pads were dissected and weighed, and an aliquot of the right retroperitoneal fat pad was used to measure adipocyte size and number. The results showed that body weight and adipocyte size (but not adipocyte number) were significantly smaller in the MCT-fed compared to the LCT-fed animals. Resting as well as maximal NE-stimulated oxygen consumption values were significantly higher in the MCT-fed than the LCT-fed rats. It is concluded that the enhanced thermogenesis induced by MCT persists despite the absence of IBAT and that the phenomenon is likely related to more extensive oxidation of MCT- in contrast to LCT-derived fatty acids, thus leading to increased oxygen consumption, enhanced dissipation of energy as heat and diminished efficiency of weight gain and deposition of body fat.