Functional screening of a metagenomic library for genes involved in microbial degradation of aromatic compounds

A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive clones. Based on their substrate specificity for various catecholic compounds, 38 clones were subjected to sequence analysis. Each insert contained at least one EDO gene, and a total of 43 EDO genes were identified. More than half of these belonged to new EDO subfamilies: I.1.C (2 clones), I.2.G (20 clones), I.3.M (2 clones) and I.3.N (1 clone). The fact that novel I.2.G family genes were over-represented in these clones suggested that these genes play a specific role in environmental aromatic degradation. The I.2.G clones were further classified into six groups based on single-nucleotide polymorphisms (SNPs). Based on the combination of the SNPs, the evolutionary lineage of the genes was reconstructed; further, taking the activities of the clones into account, potential adaptive mutations were identified. The metagenomic approach was thus used to retrieve novel EDO genes as well as to gain insights into the gene evolution of EDOs.     

Rapid induction and disappearance of electron-dense organelles following sulfide exposure in the marine annelid Branchioasychis americana

Abstract. Electron-dense organelles (EDOs) are a characteristic feature of marine annelids adapted to hydrogen sulfide, but little is known about their origin or fate or the mechanism by which they are formed. In this study, transmission electron microscopy was used to determine the appearance, quantity, and size distribution of EDOs in body wall epithelium of the sulfide-tolerant annelid Branchioasychis americana . EDOs were enclosed by either one or two lipid bilayers with a typical diameter in the range of 0.4–1.2 μm. The contents included amorphous electron-dense material, lamellated membranes, and distinct membrane-bounded structures. In animals fixed immediately after collection from the mudflat, the EDO quantity was 0.148 μm⁻². This decreased by one-third to 0.100 μm⁻² after 2 weeks under sulfide-free conditions. Subsequent exposure to 250–400 μmol L⁻¹ sulfide for ≤24 h increased the EDO quantity by 2.4-fold to 0.240 μm⁻², which reverted to control levels after 24 h under sulfide-free conditions. The results show that EDOs are transient structures that are rapidly induced by sulfide, which is consistent with the hypothesis that sulfide-induced EDOs represent the upregulated autophagic degradation of mitochondria or other cellular constituents damaged by sulfide exposure.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

ATPase activity associated with vacuoles and tonoplast vesicles isolated from the CAM plant, Kalanchoë daigremontiana

Intact vacuoles were isolated from leaves of the CAM plant, Kalanchoë daigremontiana Hamet et Perr. Both ATPase and acid phosphatase activities were found in the vacuoles. Purified tonoplast vesicles showed only ATPase activity with a pH optimum of 8.0. This activity was Mg²⁺-dependent and KCI or NaCI caused a further stimulation. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol and quercetin inhibited the ATPase almost completely at concentrations well below 1 m M. NaVo₃, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, oligomycin and NaN₃ had little or no effect. Carbonyl cyanide m -chlorophenylhydrazone stimulated the ATPase about 40% at 5 × 10⁻⁴ M. The K_m for ATP was found to be 0.55 m M. These results indicate that the ATPase found in the tonoplast membrane of Kalanchoë daigremontiana is qualitatively similar to that of other plant species.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

The impact of nitrogen supply on the potential response of a noxious, invasive weed, Canada thistle (Cirsium arvense) to recent increases in atmospheric carbon dioxide

A recognized invasive weed, Canada thistle ( Cirsium arvense L. Scop.) was grown at ambient and pre-ambient concentrations of atmospheric carbon dioxide [CO₂] (373 and 287 μmol mol⁻¹, respectively) at three levels of supplemental nitrogen (N) (3, 6 and 14.5 m M ) from seeding until flowering [77 days after sowing (DAS)]. The primary objective of the study was to determine if N supply limited the potential photosynthetic and growth response of this species to the increase in atmospheric [CO₂] which occurred during the 20th century (i.e. approximately 290 to 370 μmol mol⁻¹ CO₂). Leaf photosynthesis increased both as a function of growth [CO₂] and N supply during the first 46 DAS. Although by 46 DAS photosynthetic acclimation was observed relative to a common measurement CO₂ concentration, there was no interaction with N supply. Both [CO₂] and N increased biomass, relative growth rates and leaf area whereas root : shoot ratio was increased by CO₂ and decreased by increasing N; however, N supply did not effect the relative response to [CO₂] for any measured vegetative parameter up to 77 DAS. Due to the relative stimulation of shoot biomass, total above-ground N increased at elevated [CO₂] for all levels of supplemental N, but nitrogen use efficiency (NUE) did not differ as a function of [CO₂]. Overall, these data suggest that any potential response to increased atmospheric [CO₂] in recent decades for this noxious weedy species was probably not limited by nitrogen supply.     

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity ( K _m, 2.6 μM) and, although it also binds betaine ( K _m, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX ( K _m, 24 μM) and the betaine-specific SBP BetX ( K _m, 0.6 μM). Unlike most ABC transporter loci, caiX , betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.     

Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca²⁺-dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE-cellulose, Sephadex G-200, and calmodulin-affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a K _a value of 16.7 n m . The K _a values of peaks I and II for Ca²⁺ were 2.4 and 1.6 μ m , respectively.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Field-based measurements of oxygen uptake and swimming performance with adult Pacific salmon using a mobile respirometer swim tunnel

Novel field measurements of critical swimming speed ( U _crit) and oxygen uptake (  M o₂) in three species of adult Pacific salmon Oncorhynchus spp. up to 3·5 kg in body mass were made using two newly designed, mobile Brett-type swim tunnel respirometers sited at a number of field locations in British Columbia, Canada. Measurements of U _crit, which ranged from 1· 68 to 2·17 body lengths s⁻¹, and maximum M o₂, which ranged from 8·74 to 12·63 mg O₂ kg⁻¹ min⁻¹ depending on the species and field location, were judged to be of similar quality when compared with available data for laboratory-based studies. Therefore high quality respirometry studies were possible in the field using adult wild swimming salmonids. In addition, the recovery of wild adult Pacific salmon from the exhaustive U _crit swim test was sufficiently rapid that swimming performance could be repeated with <1 h of recovery time between the termination of the initial swim test and the start of the second test. Moreover, this repeat swimming performance was possible without routine M o₂ being reestablished. This result suggests that wild adult salmon are capable of carrying a moderate excess post-exercise oxygen consumption without adversely affecting U _crit, maximum M o₂ or swimming economy. Such capabilities may be extremely important for timely migratory passages when salmonids face repetitive hydraulic challenges on their upstream migration.     

The utilization of patchy thermal microhabitats by the ectothermic insect predator, Cicindela sexguttata

Abstract. 1. Tiger beetles ( Cicindela ) of open habitats have served as model ectotherms in studies of the dependence of activity and habitat utilization on temperature. Potential departures from the cicindelid model were investigated in Cicindela sexguttata , a species inhabiting forests where thermal resources are patchy and ephemeral.
2. Body temperatures (T_b) were determined by inserting thermocouples into beetles immediately after observing specific behaviours in the field. Cicindela sexguttata elevated T_b by basking, foraged at a preferred T_b of 32.8 °C, and stilted, sun-faced, or sought shade when T_b exceeded 35 °C. Although these behaviours were typical of Cicindela , their set points were lower than those of species in more open habitats.
3. Illuminated substrates were utilized for basking and foraging. Beetles were dispersed throughout the forest floor in early spring, but became aggregated in light gaps when the canopy leafed out. Operative temperatures of thermal models indicated that beetles were unable to maintain the preferred T_b in shade and would not be able to maintain a preferred T_b in light gaps during the autumn, when adults are normally in diapause.
4. Beetles were confined to foraging in light gaps once the canopy was closed. Foraging rate and searching speed were independent of T_b, in contrast to other cicindelids. Adults rarely searched for prey, but ambushed small arthropods that alighted in the light gap. Dependence on patches of illumination as thermal resources may increase prey capture, intraspecific encounters, and risk of predation, and preclude foraging in the autumn when Cicindela species of open habitats are also active.     

Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley ( Hordeum vulgare L.), we reported previously the existence of two BADH genes ( BBD1 and BBD2 ) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K_m of 18.9 μ M and 19.9 m M , respectively. In addition, V_max/K_m with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of ω-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4- N -trimethylaminobutyraldehyde and 3- N -trimethylaminopropionaldehyde.     

Development of Polyclonal Anti-D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [¹²⁵I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [¹²⁵I]-NCQ 298 bound reversibly and with high affinity (K_D= 0.14 n M ) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [¹²⁵I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to inhibit the high-affinity binding of the agonist N -propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Effects of waterborne complexing agents on silver uptake and depuration in rainbow trout

Rainbow trout Oncorhynchus mykiss ( c . 60 g) were exposed for 1 week to 0·1 μM silver as AgNO₃ in ion poor water (Ca c . 150 μM, pH c . 8, water temperature 13° C) with or without waterborne organic matter (27 mg C l⁻¹ as Aldrich humic acid), thiosulphate (5 μM Na₂S₂O₃) or chloride (4 mM KCl). Organic matter decreased Ag accumulation by the gills initially, but did not decrease Ag accumulation by plasma or liver. Thiosulphate decreased the amount of Ag accumulated by the gills for the entire 1 week exposure but had no effect on Ag concentrations in the plasma, liver or bile. Chloride had no effect on Ag uptake in any of the tissues examined. All three complexing agents reduced the decreases in plasma Na and Cl concentrations caused by Ag. To study the effects of waterborne complexing agents on Ag depuration, rainbow trout were exposed to 0·1 μM AgNO₃ for 1 week then placed for 8 days in Ag‐free, ion poor water with or without waterborne organic matter (55 mg C l⁻¹) or thiosulphate (5 μM). These complexing agents did not alter depuration of Ag from the gills, plasma, liver or bile. Thus, once Ag has entered a fish, subsequent elimination of internal Ag is not affected by external complexing agents.