Purification and Properties of Succinyl-CoA:3-Oxo-Acid CoA-Transferase from Rat Brain

Abstract: Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-Oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 m M acetoacetate. Apparent K_m, values were 70 μM for acetoacetate and 156 μ M for succinyl-CoA (the forward reaction), and 59 μ M for acetoacetyl-CoA and 25 m M for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

A Comparison of the Effects of Ethanol and the Competitive Glycine Antagonist 7-Chlorokynurenic Acid on N-Methyl-d-Aspartic Acid-Induced Neurotransmitter Release from Rat Hippocampal Slices

Abstract: N -Methyl- d -aspartate (NMDA; 500 μ M ) stimulated the net release of preloaded tritiated norepinephrine from rat hippocampal slices. Both ethanol and the competitive glycine antagonist 7-chlorokynurenic acid (7-CK) dose-dependently inhibited NMDA-stimulated release without affecting basal, nonstimulated efflux. These inhibitory effects were readily reversed upon washout of the drugs. Over the concentration range tested (25–200 m M ), ethanol inhibited ∼65% of NMDA-stimulated release with an estimated IC₅₀ of ∼70 m M . In contrast, 7-CK fully inhibited release (>95%) at a concentration of 30 μ M with half-maximal inhibition occurring at ∼2 μ M . The combination of 7-CK (1–30 μ M ) and ethanol (25–100 m M ) had an additive inhibitory effect on NMDA-stimulated release but did not alter the inhibitory potency of 7-CK. Calculated IC₅₀values for 7-CK in the presence of 25, 50, or 100 m M ethanol were (mean × SEM; μ M ) 2.33 (0.11), 2.38 (0.23), and 1.99 (0.30), respectively. 7-CK (3 μ M ) inhibited NMDA-stimulated [³H]norepinephrine release by ∼50%. This inhibition was fully attenuated by the addition of the glycine agonistserine with complete reversal occurring at 30 μ M d -serine. Increasing the 7-CK concentration to 10 μ M shifted the d -serine dose-effect curve to the right in a parallel fashion as expected for a competitive antagonist. In contrast, the inhibitory effects of ethanol or the combination of 7-CK (3 μ M ) and ethanol (25 or 50 m M ) were not reversed by the addition of d -serine (0.1–1,000 μ M ). Together, these results suggest that ethanol's inhibition of NMDA-stimulated [³H]norepinephrine release from hippocampal slices is not due to a simple competitive interaction with the glycine site on the NMDA receptor.     

Influence of copper on proton efflux from Saccharomyces cerevisiae and the protective effect of calcium and magnesium

Abstract The inhibitory effect of Cu on glucose-dependent H⁺ efflux from Saccharomyces cerevisiae was manifest at low (micromolar) concentrations, with the time period between the addition of glucose and commencement of H⁺ efflux, H⁺ efflux rate and duration all being affected with increasing Cu concentration (5–100 μM). Ca, at a concentration of 0.5 mM, completely removed the inhibitory effect of Cu at concentrations up to 50 μM and considerably reduced it at higher concentrations (up to 150 μM). Mg exhibited a similar but weaker protective effect against the influence of Cu. The protective effect of Ca against 50 μM Cu was evident at low Ca concentrations (2.5–5 μM), whereas Mg was effective at ≥50 μ M. In order to prevent the inhibitory effect of Cu, it was necessary to add Ca or Mg to the cell suspension before Cu addition. It is concluded that the protective effect of Ca and Mg is mediated by competitive and stabilizing interactions at the cell surface as well as physiological functions of Ca and Mg.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Influence of bicarbonate and metal ions on the development of root Fe(III) reducing capacity by Fe-deficient cucumber (Cucumis sativus) plants

The effect of bicarbonate and selected metal ions on the development of enhanced root Fe(III) reducing capacity (a response to Fe deficiency of dicotyledons) was studied in young plants of cucumber ( Cucumis sativus L. cv. Ashley) grown in nutrient solution. Pretreatment of 11-day-old Fe-deficient cucumber plants with 20 m M NaHCO₃, for at least 23 h prior to determination of root Fe(III) reducing capacity, markedly inhibited this response. The inhibitory effect of bicarbonate could be partly reversed by a 4- to 8-h treatment with either 10 μ M MnSO₄, 10 μ M FeEDDHA, 2 μ M ZnSO₄, 0.5 μ M NiCl₂, or 0.25 μ M , or CoSO₄ (final concentrations), added to the nutrient solution. By contrast, the addition of other salts of metal ions, like CuSO₄ and Cd(NO₃)₂, at 0.25, 0.5 or 1 μ M , or MgSO₄, at 0.5, 1 or 2 m M (final concentrations), had no beneficial effect. The results suggest that bicarbonate may inhibit the development of root Fe(III) reducing capacity by diminishing the availability of certain metal ions required for this response.     

PRODUCT INHIBITION OF RAT BRAIN HISTAMINE-N-METHYLTRANSFERASE

Abstract— The inhibition of S -adenosylmenthionine: histamine- N -methyltransferase (EC 2.1.1.8; HMT) by its products, 3-methylhistamine (3-MetHm) and S -adenosyl- l -homocysteine (SAH), was examined using a preparation of the enzyme which was partially purified from rat brains. SAH was found in in vitro experiments, to be a competative inhibitor of HMT in relation to S -adenosyl- l -methionine (SAM), with a K _i= 5.6 μM. SAH was shown to be a non-competitive inhibitor with respect to histamine (Hm) ( K _i= 5.0 μM). The K _m's for SAM and Hm were 10.2 and 3.0 μM respectively. On the other hand, 3-MetHm was determined to be a non-competitive inhibitor of HMT with respect to Hm ( K _i= 8.7 μM) and an uncompetitive inhibitor with respect to SAM ( K _i= 9.6 μM). These results suggest that the addition of the substrate to, and the release of products by, HMT occurs sequentially. In the nomenclature Of C leland (1963) the reaction is seemingly of the 'ordered Bi-Bi' type.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

Evidence that brassinosteroid stimulates auxin-induced ethylene synthesis in mung bean hypocotyls between S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid

Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl₂ in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene.     

Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Induction of preputium eversion by peptides, serotonin receptor antagonists, and selective serotonin reuptake inhibitors in Biomphalaria glabrata

Abstract. Eversion of the preputium is one of the initial steps in the male copulatory behavior of freshwater pulmonates. Previous experiments have shown that serotonergic mechanisms are involved in eversion in the snail Biomphalaria glabrata because the vertebrate 5-HT₁ receptor antagonist methiothepin caused long-lasting eversion in a dose-dependent manner. In this study, we tested a variety of serotonergic receptor ligands, bioactive peptides, and selective serotonin reuptake inhibitors (SSRIs) for their ability to induce preputium eversion in B . glabrata in order to elucidate the physiological mechanism of eversion. Of 15 compounds tested, five significantly induced preputium eversion: the serotonin receptor antagonists methiothepin (1 and 10 μM; p<0.0001), cyproheptadine (1–10 μM; p<0.007–0.0001), and mianserin (5–50 μM; p<0.01–0.001), the molluscan cardioactive peptide FMRFamide (10 and 50 μM; p<0.0002–0.0001), and the SSRI fluoxetine (=Prozac, 10–100 μM; p<0.0003–0.0001). Serotonin itself neither induced eversion nor blocked methiothepin-induced eversion. This suggests that fluoxetine is not acting as an SSRI, but potentially as a receptor ligand. These preliminary data shed light on the possible physiological mechanism of preputium eversion in B . glabrata and suggest similarity with that of the model freshwater gastropod Lymnaea stagnalis .     

Reaction of Muscimol with 4-Aminobutyrate Aminotransferase

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA-T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, K _m (app) = 1.92 ± 0.24 m M , specific activity = 7.33 ± 0.27 μmol/min/mg ( k _cat= 13.7s⁻¹), and for muscimol, K _m (app) = 1.27 ± 0.15 m M , specific activity = 0.101 ± 0.009 μmol/min/mg ( k _cat= 0.19s⁻¹). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first-order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K = 1.1 ±0.18 m M and k _max= 0.065 ± 0.004 s⁻¹ (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA-T.     

Abscisic acid induced stress-like polyamine pattern in wheat seedlings, and its reversal by potassium ions

In 3-day-old wheat ( Triticum aestivum L. cv. Marinat) seedlings, 100 μ M ABA blocked the growth and altered the level of K⁺ in both the shoot and root. The presence of ABA increased the putrescine titer during a 24-h treatment. Increasing the endogenous level of K⁺ by the addition of 10 m M KCl to the ABA-treated seedlings, inhibited the effect of ABA on growth and putrescine level. In both tissues, ABA increased putrescine content at low concentrations (1 μ M ), reaching the maximal effect at 100 μ M . Putrescine increase induced by ABA was inhibited by both α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO) in shoots while only the inhibitor of arginine decarboxylase was effective in the root. The presence of ABA modulated, in opposite ways, ornithine and arginine decarboxylase activities. These results are discussed in relation to ion balance under stress.     

Substrates dissociate dopamine transporter oligomers

Substrate-induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N-terminal tagged DAT constructs. Dopamine (100 μM) or amphetamine (2–10 μM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10–1000 μM) and amphetamine (0.2–200 μM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline or Cu²⁺. Inhibition of endocytosis by 10 μM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 μM DA or 2 μM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT.     

Muscarine Receptors Regulating Electrically Evoked Release of Acetylcholine in Hippocampus Are Linked to Pertussis Toxin-Sensitive G Proteins but Not to Adenylate Cyclase

Abstract: [³H]Acetylcholine release elicited with 360 pulses/3 Hz from slices of rabbit hippocampus is facilitated in the presence of the muscarine (M) receptor antagonist atropine (indicating the existence of autoinhibition) and diminished by the M receptor agonists carbachol and oxotremorine. W-Ethylmaleimide (30 μM ) and pertussis toxin (8 μg/ml) counteracted antagonist-induced facilitation and agonist-induced inhibition of release, suggesting that a pertussis toxin-sensitive GTP-binding protein is involved in the chain of events mediating activation of M receptors to inhibition of release. Neither 8-bromo-cyclic AMP (300 μM ), a membrane analogue of cyclic AMP, nor rolipram (10 μM ), a phosphodiesterase inhibitor, affected electrically evoked release of [³H]acetylcholine. They also did not influence the oxotremorine-induced inhibition of transmitter release. In conclusion, no evidence was found for the assumption that activation of M autoreceptors is linked to inhibition of adenylate cyclase.     

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-μM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N -acetyl- l -cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-μM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10–15 μM, whereas, at a concentration of >20-μM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-μM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-μM curcumin protected fibroblasts against 25-μM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-μM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 μM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.     

Ethylene as a possible mediator of light-induced inhibition of root growth

Eliasson, L. and Bollmark, M. 1988. Ethylene as a possible mediator of light-induced inhibition of root growth. - Physiol. Plant. 72: 605–609.
Pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the possible role of ethylene in light-induced inhibition of root elongation. Illumination of the roots with white light inhibited root elongation by 40–50% and increased ethylene production by the roots about 4-fold. Our main approach was to use exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), supplied in the growth solution, to monitor ethylene production of the roots independent of light treatment. Ethylene production of excised root tips increased with increasing ACC concentrations. The rate of ethylene production in dark-grown roots treated with 0.1 μ M ACC was similar to that caused by illumination. Low ACC concentrations (0.01–0.1 μ M ) decreased the rate of root elongation, especially in seedlings grown in the dark, and 0.1 μ M ACC inhibited elongation to about the same extent as light. In light the roots curved and grew partly plagiogravitropically. This effect was also simulated by the 0.1 μ M ACC treatment. At 1 μ M and higher concentrations, ACC inhibited root growth almost completely and caused conspicuous curvatures of the root tips both in light and darkness. Inhibitors of ethylene synthesis and action partially counteracted the inhibition of root elongation caused by light. These observations suggest that the increase in ethylene production caused by light is at least partly responsible for the decreased growth of light-exposed roots.     

Influence of phytohormones and other effectors on proton extrusion by isolated protoplasts from rape leaves

The roles of phytohormones and fusicoccin in H⁺ extrusion by isolated protoplasts from rape leaves ( Brassica napus L. cv. Belinda) were investigated and compared to results obtained with leaf segments of the same plants. Net H⁺ release by protoplasts, which was at least partly due to ATPase activity, was enhanced by 10 μ M indole-3-acetic acid and reduced by 20 μ M abscisic acid, whereas fusicoccin (10 μ M ), brassinosteroid (3 μ M ), kinetin (20 μ M ) and gibberellic acid (10 μ M ) had no effect. Hormone effects and H⁺ release were not detectable with leaf segments from the same plants. However, using field-grown plants, indole-3-acetic acid and especially fusicoccin stimulated the acidification of the external medium by leaf segments. Hormonecontrolled H⁺ release by leaf cells is interpreted as the first step in acid-triggered and turgor-regulated cell growth.     

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.