The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.     

Diel variation in ammonia excretion,glutamine levels,and hydration status in two species of terrestrial isopods

Terrestrial isopods (suborder Oniscidea) excrete most nitrogen diurnally as volatile ammonia, and ammonia-loaded animals accumulate nonessential amino acids, which may constitute the major nocturnal nitrogen pool. This study explored the relationship between ammonia excretion, glutamine storage/mobilization, and water balance, in two sympatric species Ligidium lapetum (section Diplocheta), a hygric species; and Armadillidium vulgare (Section Crinocheta), a xeric species capable of water-vapor absorption (WVA). Ammonia excretion (12-h), tissue glutamine levels, and water contents were measured following field collection of animals at dusk and dawn. In both species, diurnal ammonia excretion exceeded nocturnal excretion four- to fivefold while glutamine levels increased four- to sevenfold during the night. Most glutamine was accumulated in the somatic tissues (body wall). While data support the role of glutamine in nocturnal nitrogen storage, potential nitrogen mobilization from glutamine breakdown (162 mol g^–1 in A. vulgare) exceeds measured ammonia excretion (2.5 mol g^–1) over 60-fold. This may serve to generate the high hemolymph ammonia concentrations seen during volatilization. The energetic cost of ammonia volatilization is discussed in the light of these findings. Mean water contents were similar at dusk and dawn in both species, indicating that diel cycles of water depletion and replenishment were not occurring.     

Energy coupling and respiration in Nitrosomonas europaea.

Intact cells of Nitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. The Km value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of "endogenous" substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.     

Ethylene oxidation by Nitrosomonas europaea

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 mol h^-1 mg protein^-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic V _max. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate K _i of 80 M was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a k _cat/K _m value for ethylene of 1.1 relative to NH ₄ ⁺ , or 0.04 relative to the true natural substrate, NH₃. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.     

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 mol· h^-1·mg protein^-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k _cat/K _m for benzene relative to k _cat/K _m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH ₄ ⁺ · mg chlorophyll (Chl)^-1, with an average production rate of 14 mol NH ₄ ⁺ · mg Chl^-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH ₄ ⁺ · mg Chl^-1 and 6.7 mol NH ₄ ⁺ · mg Chl^-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Sensory deficits induced by cadmium in banded kokopu, Galaxias fasciatus, juveniles

The potential for cadmium to produce sensory deficits in the mechanosensory lateral line and olfactory systems was examined in migratory Galaxias fasciatus juveniles or whitebait. Using a two-choice chamber apparatus, three groups of whitebait were tested for a known attraction to adult pheromones and then exposed to either 0.1, 0.5 or 1g Cd⁺² l^-1 for 48h and retested. The attraction to adult pheromones had been eliminated after exposure to concentrations of 0.5 and 1g Cd⁺² l^-1, indicating these levels of cadmium exposure had impaired olfactory function. Rheotaxis trials were conducted to determine the level of cadmium exposure which would inhibit lateral line function. The lateral line system was not blocked until a concentration of 2g Cd⁺² l^-1. The blocking of the lateral line and olfactory sensory systems was reversible. After 14 days recovery in clean freshwater both rheotaxis and the attraction to adult pheromones had returned. Whitebait were also tested for a preference/avoidance response at 2g Cd⁺² l^-1. Fish showed neither a preference for, or an avoidance of, a concentration which would disable both the lateral line and olfactory sensory systems. The disabling of these sensory systems may render migratory cues undetectable, affecting habitat selection by whitebait, which may ultimately affect the distribution of banded kokopu populations.     

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the K_m value was higher (70 M) than for net influx (24 M); the exchange efflux is proposed to be mediated by the same transport system with a K_m value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H⁺ per sugar; during the onset of hexose transport there was a K⁺ exit of 0.94 K⁺ per sugar for charge compensation. (It was assumed that the real stoichiometries are 1 H⁺ and 1 K⁺ per sugar.) The K_m values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.Abbreviations 3-OMG 3-O methylglucose - DMO 5,5-dimethyl-2, 4-oxazolidinedione - TPP tetraphenylphosphonium chloride - CCCP carbonyl cyanide, m-chlorophenylhydrozane     

Genetic regulation of theAchaete-scute complex ofDrosophila melanogaster

Summary Mutants in two loci,hairy (h ⁺) andextramacrochaetae (emc ⁺), produce phenotypes corresponding to an excess of function of theachaete-scute complex (AS-C), that is, they cause the appearance of extra chaetae. These mutants, although recessive in normal flies, become dominant in the presence of extra doses of AS-C. Here we study the interactions between these three genes, in an attempt to elucidate their relationships. The results show that the insufficiency produced byh oremc mutants can be titrated by altering the number of copies of AS-C. Moreover, excess of function of AS-C produced by derepression mutants within the complex (Hairy-wing) can also be titrated by altering the number of wild type copies of⁺ oremc ⁺. These specific interactions indicate that bothh ⁺ andemc ⁺ code for repressors of AS-C that interact with theachaete andscute region of the complex respectively.     

Transfer of defined numbers of chloroplasts into albino protoplasts by subprotoplast/protoplast microfusion: chloroplasts can be “cloned”, by using suitable plastome combinations or selective pressure

Summary Defined numbers (1–5) of (donor) chloroplasts were transferred into (acceptor) protoplasts of plastid albino mutants by subprotoplast/protoplast microfusion. Single transferred plastids gave rise to new organelle populations in the progeny of the fusion products when suitable combinations of plastomes were used or when selective pressure for the plastome transferred was applied. This process is termed chloroplast cloning and is the first reported case of cloning a cell organelle. The plastome combination and the presence or absence of selective pressure were found to influence the frequencies with which cell lines, containing both plastomes or acceptor or donor only, were obtained, and the number of cell generations needed for complete segregation — as measured by the duration of culture before the green donor plastome could be detected. The high frequency of cell lines and regenerated shoots recovered with donor plastome only, even when only a single chloroplast was transferred, leads to the conclusion that all organelles present in the fusion product contribute to the organelle population of the progeny, i.e. organelle death or loss are not regularly occurring events during plant regeneration from protoplasts in Nicotiana tabacum.Some of the results reported here were presented at the 8th International Protoplast Symposium, Uppsala 1991     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.