Manganese toxicity towards Saccharomyces cerevisiae : Dependence on intracellular and extracellular magnesium concentrations

Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn²⁺ or higher, but a tolerance to lower Mn²⁺ concentrations was observed. The inhibitory effects of 2.0 mM Mn²⁺ were eliminated by supplementing the medium with excess Mg²⁺ (10 mM), whereas addition of excess Ca²⁺ and K⁺ had negligible effect on Mn²⁺ toxicity. Growth inhibition by Mn²⁺, in the absence of a Mg²⁺ supplement, was attributed to Mn²⁺ accumulation to toxic intracellular levels. Mn levels in S. cerevisiae grown in Mg²⁺-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium. Mn²⁺ toxicity was also influenced by intracellular Mg, as Mn²⁺ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone. Cells with low intracellular levels of Mg were more susceptible to Mn²⁺ toxicity than cells with high cellular Mg, even when sequestered Mn²⁺ levels were similar. A critical Mg:Mn ratio of 2.0 was identified below which Mn²⁺ toxicity became acute. The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn²⁺. Received: 18 June 1997 / Received last revision: 10 January 1998 / Accepted: 24 January 1998     

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA. Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Active calcium ion transport in Xenopuslaevis skin

The skin of intact, free-swimming Xenopus laevis transports Ca²⁺ inwardly in a manner that is proportional to the external [Ca²⁺] up to about 0.3 mmol · l⁻¹, saturates above 0.3 mmol · l⁻¹, and is opposed to the electrochemical gradient. Efflux is relatively constant at external concentrations between 0.016 and 0.6 mmol · l⁻¹; net flux which is negative below 0.125 mmol · l⁻¹ becomes positive above this external [Ca²⁺]. Allometric analysis suggests that both Ca²⁺ influx and efflux scale to the 2/3 power approximately like surface area. There were no significant differences in influx between summer and fall animals; however, efflux was greater in the fall and this resulted in a change from positive balance in the summer to negative balance in the fall. Isolated skins were shown to support a Ca²⁺ uptake rate of nearly 30 nmol · cm⁻² · h⁻¹. The phenylalkylamine verapamil in the apical bathing solution significantly inhibited this at 25 μmol · l⁻¹. The benzothiazepine diltiazem was also effective at 50 μmol · l⁻¹ while the dihydropyradine nifedipine was ineffective up to 100 μmol · l⁻¹. The inorganic ion La³⁺ was effective at blocking Ca²⁺ uptake at 300 μmol · l⁻¹; Ni²⁺ was also effective at 500 μmol · l⁻¹ but Co²⁺ was ineffective up to 500 μmol · l⁻¹. These results suggest that apical calcium channels in Xenopuslaevis skin have properties similar to mammalian L-channels and fish gill Ca²⁺ channels. Accepted: 23 January 1997     

High volumetric yields of functional dimeric miniantibodies in Escherichia coli,using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl⁻¹ in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-termi-nal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hok/sok suicide system, improving plasmid maintenance, and the inducible lac p/o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l⁻¹ after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fed-batch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 g l⁻¹, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions. Received: 26 February 1996 / Received revision: 1 August 1996 / Accepted: 12 August 1996     

Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand

The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd³⁺). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd³⁺, which ranged from 350 μmol g⁻¹ for B. subtilis to 5.1 μmol g⁻¹ for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension. Received: 8 November 1999 / Received revision: 3 February 2000 / Accepted: 4 February 2000     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export. Received: 9 January 1998 / Accepted: 4 May 1998     

Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp.

The marine photosynthetic bacterium Chromatium sp. successfully removed orthophosphate when grown phototrophically. The phosphate-uptake rate was almost constant at more than 5.0 mg- PO₄ ³⁻/l in synthetic medium. Addition of seawater causes flocculation of this strain. The successful use of seawater as an inexpensive source of magnesium could prove to be effective in the removal of photosynthetic bacterial cells from a medium. A semicontinuous culture system was used for the removal of low concentrations of phosphate and the phosphate-uptake activity of Chromatium sp. was maintained under 0.1 day⁻¹ dilution rate. This strain was also able to remove high concentrations of phosphate from domestic sewage. Received 24 May 1996 / Received revision: 5 August 1996 / Accepted: 6 September 1996     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996     

The use of amiloride to uncouple branchial sodium and proton fluxes in the brown trout, Salmo trutta

Resting proton, ammonium and sodium fluxes in Salmo trutta were 492.6 ± 19.5 (n = 29); 122.9 ± 34.2 (n = 28) and 277.1 ± 18.5 (n = 50) μmol · kg⁻¹ · h⁻¹, respectively. The resting transepithelial potential was found to be composed of three successive potentials, the outermost averaging −7.36 ± 0.19mV, the second, −14.3 ± 1.4 mV and the third −37 ± 1.7 mV. Amiloride inhibits the proton, ammonium and sodium fluxes in a dose-dependent manner at concentrations of 0.5 mmol · 1⁻¹ and 0.1 mmol · l⁻¹, but at 0.01 mmol · l⁻¹, proton and ammonium fluxes remained at control levels whilst the sodium was reduced to 70.59 ± 7.29 μmol · kg⁻¹ · h⁻¹. The trans-epithelial potential was effected in a bi-phasic manner by 0.5 mmol · l⁻¹ amiloride. An initial hyperpolarisation of ca. 6 mV was followed by a sustained depolarisation of ca. 14 mV (towards zero) which persisted until the amiloride was washed off the gill. The initial hyperpolarisation was thought to reflect a rapid inhibition of a positive inward sodium current and the subsequent depolarisation was due to the inhibition of a positive outward current (proton) which would abolish the transepithelial potential. However, at 0.01 mmol ·  l⁻¹ only the hyperpolarisation was seen, due to the inhibition of only the inward sodium current. Acetazolamide (0.1 mmol · l⁻¹) was found to have no significant effect on the proton, ammonium and sodium fluxes. These results indicate that the proton and sodium fluxes across the gill of the freshwater trout are not tightly linked. While this suggests that the trout gill resembles the model of Ehrenburg et al. (1985) of sodium uptake in frog skin, the apical potentials measured in the pavement epithelial cell(s) are too low to account for sodium uptake unless the activity of the sodium in the cells is very low. Accepted: 8 August 1996     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

Water and sodium balances and metabolic physiology of house mice (Mus domesticus) and short-tailed mice (Leggadina lakedownensis) under laboratory conditions

A laboratory study investigated the metabolic physiology, and response to variable periods of water and sodium supply, of two arid-zone rodents, the house mouse (Mus domesticus) and the Lakeland Downs short-tailed mouse (Leggadina lakedownensis) under controlled conditions. Fractional water fluxes for M. domesticus (24 ± 0.8%) were significantly higher than those of L. lakedownensis (17 ± 0.7%) when provided with food ad libitum. In addition, the amount of water produced by M. domesticus and by L. lakedownensis from metabolic processes (1.3 ± 0.4 ml · day⁻¹ and 1.2 ± 0.4 ml · day⁻¹, respectively) was insufficient to provide them with their minimum water requirement (1.4 ± 0.2 ml · day⁻¹ and 2.0 ± 0.3 ml · day⁻¹, respectively). For both species of rodent, evaporative water loss was lowest at 25 °C, but remained significantly higher in M. domesticus (1.1 ± 0.1 mg H₂O · g^−0.122 · h⁻¹) than in L. lakedownensis (0.6 ± 0.1 mg H₂O · g^−0.122 · h⁻¹). When deprived of drinking water, mice of both species initially lost body mass, but regained it within 18 days following an increase in the amount of seed consumed. Both species were capable of drinking water of variable saline concentrations up to 1 mol · l⁻¹, and compensated for the increased sodium in the water by excreting more urine to remove the sodium. Basal metabolic rate was significantly higher in M. domesticus (3.3 ± 0.2 mg O₂ · g^−0.75 · h⁻¹) than in L. lakedownensis (2.5 ± 0.1 mg O₂ · g^−0.75 · h⁻¹). The study provides good evidence that water flux differences between M. domesticus and L. lakedownensis in the field are due to a requirement for more water in M. domesticus to meet their physiological and metabolic demands. Sodium fluxes were lower than those observed in free-ranging mice, whose relatively high sodium fluxes may reflect sodium associated with available food. Accepted: 16 August 1999     

Ca2+ regulation of heart contractility in Octopus

Isometric force development of electrically paced preparations isolated from the systemic heart of Octopus vulgaris were utilized to examine the regulation of contractility by Ca²⁺. Increases in extracellular Ca²⁺, to the physiological level, resulted in enhancement of twitch force. For instance, at 36 beats · min⁻¹ an increase in Ca²⁺ from 3 to 9 mmol · l⁻¹ resulted in a threefold increase in twitch force development. When steady-state contraction at 12 beats · min⁻¹ was followed by a rest period of either 5 or 10 min, the first contraction always exhibited either an increase in twitch force or stayed unchanged such that post-rest twitch force was about 133% of the last value in the steady-state train. Ryanodine (12.5 μmol · l⁻¹), which is considered to be a specific inhibitor of the Ca²⁺ storage and release capabilities of the sarcoplasmic reticulum (SR), was applied to further assess Ca²⁺ handling. Twitch force fell to about 22% of the preteatment level in preparations paced at either 12 or 36 beats · min⁻¹. In all preparations the frequency transition from 12 to 36 beats · min⁻¹ was associated with an increase in resting tension. The␣increase␣was 37 ± 14% prior to ryanodine treatment and was significantly elevated to 127 ± 33% following treatment. When steady-state contraction at 36 beats · min⁻¹ was followed by a rest period of 10 s, the first contraction was not significantly different from the last beat in the train prior to ryanodine; however, with ryanodine treatment, post-rest twitch force development significantly decreased. Twitch force development was regular at pacing rates of up to 300 beats · min⁻¹. Twitch force was maintained up to rates of 84 beats · min⁻¹ but␣decreased thereafter and reached a value of about 10% at 300 beats · min⁻¹. Resting tension increased substantially as frequency was elevated from 12 to 36 beats · min⁻¹ and then gradually increased as frequency was further elevated to 180 beats · min⁻¹. In conclusion, the Octopus ventricle is dependent upon extracellular Ca²⁺ for contraction. A post-rest potentiation of force development, the negative impact of ryanodine, and the ability to respond regularly at high pacing rates imply a strong reliance on the SR in Ca²⁺ cycling based on criteria established for vertebrate hearts. Accepted: 19 January 1997     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Lactobacillus sanfranciscensis CB1: manganese, oxygen, superoxide dismutase and metabolism

The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn²⁺. Mn²⁺ was especially related to the highest substrate consumption. Aerobiosis and Mn²⁺ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H₂O₂ meant that the survival of air-grown cells supplied with Mn²⁺ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn²⁺ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H₂O₂ treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1. Received: 24 July 1998 / Received last revision: 11 November 1998 / Accepted: 13 November 1998     

Insulin and glucagon immunoreactivity during high-intensity exercise under opiate blockade

Eight fit men [maximum oxygen consumption (O_2max) 64.6 (1.9) ml · kg⁻¹ · min⁻¹, aged 28.3 (1.7) years (SE in parentheses) were studied during two treadmill exercise trials to determine the effect of endogenous opioids on insulin and glucagon immunoreactivity during intense exercise (80% O_2max). A double-blind experimental design was used with subjects undertaking the two exercise trials in counterbalanced order. Exercise trials were 20 min in duration and were conducted 7 days apart. One exercise trial was undertaken following administration of naloxone (N; 1.2 mg; 3 ml) and the other after receiving a placebo (P; 0.9% NaCl saline; 3 ml). Prior to each experimental trial a flexible catheter was placed into an antecubital vein and baseline blood samples were collected. Immediately after, each subject received either a N or P bolus injection. Blood samples were also collected after 20 min of continuous exercise (running). Glucagon was higher (P < 0.05), while insulin was lower (P < 0.05), during exercise compared with pre-exercise values in both trials. However, glucagon was higher (P < 0.05) in the P than in the N exercise trial [141.4 (8.3) ng · l⁻¹ vs 127.2 (7.6) ng · l⁻¹]. There were no differences in insulin during exercise between the P and N trials [50.2 (4.3) pmol · l⁻¹ vs 43.8 (5) pmol · l⁻¹]. These data suggest that endogenous opioids may augment the glucagon response during intense exercise. Accepted: 15 June 1996     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996