EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE ACCUMULATION OF ASTAXANTHIN IN CHEMOSTAT CULTURES OF HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)

The effect of dissolved oxygen partial pressure on the accumulation of astaxanthin in the green alga Haematococcus lacustris ( Gir.) Rostaf (UTEX16) was studied in N-limited continuous chemostat cultures. The steady-state astaxanthin content measured against culture volume, cell number, and biomass dry weigh of Haematococcus cultures was proportional to the dissolved O₂ partial pressure in the culture medium, over the range of 0–50% O₂ The steady-state biomass dry weight concentrations remained at between 0.52 and 0.57 g. L^-1 over the range of dissolved O₂ partial pressure studied. Steady-state cell densities at dissolved O₂ partial pressures above the air saturation level (1.13–1.58 × 10⁵ cells.mL^-1) were about half of that measured at lower dissolved O₂ partial pressures (2.42–2.63 × 10⁵ cells.mL^-1). Both biflagellated zoospores and nonmotile aplanospores were found at steady state. The fraction of nonmotile cells was higher at dissolved O₂ partial pressures above the air saturation level (94.44–98.01%) than at dissolved O₂ partial pressure below the air level (79.64–86.12 and 91.75% ).     

THE UPTAKE OF [3H]CHOLINE BY SNAIL (HELIX POMATIA) NERVOUS TISSUE IN VITRO

Abstract— When suboesophageal ganglia of the snail Helix comalia were incubated at 25°C in a medium containing [³H]choline, tissue: medium ratios of about 14:1 were obtained after 20 min incubation, and only 15°, of the accumulated choline was metabolized to form [³H]acetylcholine. The uptake of [³H]choline showed saturation kinetics and was dependent upon temperature and sodium ions. Kinetic analysis suggested the existence of a high affinity uptake process (K_m= 1.7 μM, V_max= 0.21 nmol/g/min) and a low affinity process (K_m= 100 μM, V_max= 1.2 nmol/g/min). The high affinity uptake differed from the low affinity system in that it was sensitive to various metabolic inhibitors and was competitively inhibited by low concentrations of hemicholinium- and acetylcholine. Neither uptake system was greatly influenced by the absence of calcium, potassium or magnesium ions or by the presence of low concentrations of 5-HT, dopamine. tetrabenazine, chlorpromazine, decamethonium, nalaxone or imipramine. The high affinity uptake process may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Changes in ethanol, lactate and malate content in Acer pseudoplatanus cells: effects of fusicoccin and O2 availability

Changes in the contents of ethanol, lactate and malate were determined at different activities of the plasma membrane H⁺ pump [in the presence and absence of fusicoccin (FC)] and at different O₂ availability in cultured cells of Acer pseudoplatanus L. FC induced acidification of the medium under all tested conditions of O₂ availability. At low O₂ concentrations both ethanolic and lactic fermentations occurred, and FC markedly stimulated lactate production but had no effect on ethanol production. There was also a small, stimulating effect of FC on malate production. At high O₂ concentrations no ethanol production was observed and lactate production was reduced. Under these conditions the stimulating effect of FC on lactate production decreased, while that on malate production increased. FC-induced synthesis of lactate and malate is interpreted as depending on the activation of lactate dehydrogenase (EC 1.1.1.27) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) (alkaline pH optima), respectively, due to the alkalinization of the cytoplasmic pH resulting from the stimulation of the H⁺ pump by FC. These results suggest that the balance between the two pH stat systems depends on the availability of O₂.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Swimming alters responses to hypoxia in the Adriatic sturgeon Acipenser naccarii

When the Adriatic sturgeon Acipenser naccarii was exposed to progressive hypoxia under static conditions, it exhibited a linear decline in O₂ uptake, behaving as an 'oxyconformer'. When, however, it was allowed to swim at a low sustained speed, it could regulate O₂ uptake down to a mean ± s . e . critical ( P _crit) of 4·9 ± 0·5 kPa ( n = 6). At moderate levels of hypoxia, static fish exhibited significant reductions in arterial blood O₂ content, and increases in plasma lactate, which were not observed in swimming animals.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

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

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

An Investigation of the Oxidation-reduction Potential and of the Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores, using Platinum Electrodes

S ummary . The changes in electrode potential recorded by a platinum foil electrode, and in pH, during the incubation of Clostridium butyricum spores in a medium continuously sparged with a sterile gas mixture have been correlated with the biological events occurring simultaneously. The marked fall in E _h which occurs during incubation is recorded by this type of electrode as occurring during the period of rapid cell replication; this observation may be an artefact associated with foil electrodes and the authors feel that results obtained with such electrodes should be interpreted with caution. The stimulating effect of CO₂ and the increasingly inhibitory effect on germination of O₂, as the concentration of the latter in the gas mixture is increased, have been noted. The inhibitory effect is thought to be due to the O₂ itself and not to the high E _h produced in the medium by the O₂.     

Growth of methanotrophs in methane and oxygen counter gradients

Abstract A gel-stabilized system with counter gradients of CH₄ and O₂ was used to grow methanotrophs from wetland, agricultural and forest soils and lake sediment. Columns of semi-solid nitrate- or ammonium-minerai salts medium were continuously flushed at opposite ends with CH₄ and O₂ to create opposing concentration gradients of the two gases. Methanotrophs grew from all samples except forest soil, and were visible as thin bands after 5 to 15 days of incubation. The position of growth was CH₄ and O₂ concentration-dependent and occurred at the point of maximum possible CH₄ oxidation, where both substrates were completely consumed. Evidence was obtained for denitrification and nitrification activities concomitant with CH₄ oxidation. This approach may be useful to isolate methanotrophs with different CH₄ and O₂ requirements and to study their interactions with other groups of bacteria in nature.     

The presence of oxygen in rumen liquor and its effects on methanogenesis

In situ measurement of O₂ in the rumen liquor of cows, sheep and goats using a membrane-covered O₂ electrode revealed the presence of up to 1630 nmol/l O₂; O₂ became undetectable immediately after feeding of animals. The effects of O₂ on H₂ production and methanogenesis in samples of rumen liquor were investigated using a mass spectrometer fitted with a membrane inlet system. Methanogenesis was totally and irreversibly inhibited after short term exposure (about 10 min) to 5 KPa (0·05 atm) O₂; H₂ production was unaffected. Glucose additions produced rapid transient increases in H₂ levels and increased O₂ uptake.     

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1-3 h in a low O₂ atmosphere. Basal TH levels were 5-6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty-eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O₂ in N₂ for two 30-min periods (20-min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O₂ in N₂ for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.     

Nitrogen fixation (C2H2 reduction) by Medicago nodules and bacteroids under sodium chloride stress

Medicago ciliaris (L.) All., a salt-tolerant legume, was not nodulated by Rhizobium meliloti (2011), a strain commonly used for field inoculation of alfalfas. A strain of Rhizobium meliloti (ABS7) was isolated from saline Algerian soils. It is generally more salt-resistant than strain 2011, exhibits a higher rate of growth and induces the formation of nodules on M. ciliaris . C₂H₂ reduction activity of M. ciliaris nodules was inhibited by 50% in the presence of 200 m M NaCl in the culture medium. whereas 100 m M NaCl was sufficient to inhibit the activity of nodules of M. sativa (L. cv. Europe). C₂H₂ reduction by bacteroids, isolated from nodules of the two species of alfalfa, was directly inhibited by the presence of NaCl in the incubation medium. In both cases, glucose could support bacteroid nitrogen fixation, but only in a narrow range of O₂ tensions. Bacteriods from M. ciliaris were more tolerant to salt than M. sativa ones. The salt resistance of bacteroids from nodules of plants watered with NaCl solutions was not improved in either species. Salt directly added to the incubation mixture of bacteroids or to the culture medium of plants inhibited O₂ uptake of bacteroids isolated from nodules of both M. ciliaris and M. sativa . The depressive effect of NaCl on bacteroid C₂H₂ reduction could be directly related to the drop in bacteroid respiration. The nitrogen fixation capacity of the M. ciliaris-Rhizobium meliloti (ABS7) symbiosis under saline conditions leads us to recommend the introduction of this association in salt-troubled areas.     

Oxygen Consumption of Coccidial Sporozoites During in Vitro Excystation

SYNOPSIS. Oxygen consumption of sporozoites of Eimeria acervulina, E. necatrix , and E. meleagrimitis was determined during in vitro excystation. Increase in O₂ consumption occurred only when the permeability of the oocyst membrane was altered by either grinding or incubation in cystine buffer with CO₂ when excystation fluid was present. Addition of 10⁻³ M KCN completely inhibited respiration. The O₂ consumption reached a peak during the time the sporozoites were excysting, and then decreased to a lower level where it remained steady while the sporozoites were awaiting stimulus to penetrate host cells.     

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

Volatile organic compound (VOC) emissions from tobacco ( Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O₃) were investigated using proton-transfer-reaction mass-spectrometry (PTR-MS) and gas chromatography mass-spectrometry (GC-MS) to find a quantitative reference for plants' responses to O₃ stress. O₃ exposures to illuminated plants induced post-exposure VOC emission bursts. The lag time for the onset of volatile C₆ emissions produced within the octadecanoid pathway was found to be inversely proportional to O₃ uptake, or more precisely, to the O₃ flux density into the plants. In cases of short O₃ pulses of identical duration the total amount of these emitted C₆ VOC was related to the O₃ flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C₆ product was emitted per five O₃ molecules taken up by the plant. A threshold flux density of O₃ inducing emissions of C₆ products was found to be (1.6 ± 0.7) × 10⁻⁸ mol m⁻² s⁻¹.     

THE UPTAKE OF γ-[3H]AMINOBUTYRIC ACID BY SLICES FROM VARIOUS REGIONS OF RAT BRAIN AND THE EFFECT OF LITHIUM

Abstract— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [³H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity ( K_m = 1.2 ± 10^-5 M) while the other has a lower affinity ( K_m = 4 ± 10^-4 M). Studies at low GABA concentration (5 ± 10^-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation.
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.     

Photosynthesis and respiration of a diatom biofilm cultured in a new gradient growth chamber

Abstract A diatom biofilm was grown in a chamber developed for culture of biofilms in chemical gradients. The diatoms grew on a polycarbonate membrane filter which separated a sterile reservoir, with added phosphate, from a reservoir without phosphate. Within 3 weeks of inoculation, a thick biofilm developed on the surface of the filter. The biofilms were homogeneous and therefore suitable for calculations of O₂ diffusion fluxes from concentration profiles of O₂. Profiles of O₂, pH, and gross photosynthesis at different light intensities and liquid medium concentrations of dissolved inorganic carbon and O₂ were measured with microelectrodes. Respiratory activity in a layer of the biofilm was determined as the difference between gross photosynthesis and outflux of O₂ from that layer. The photosynthetic activity in a well-developed biofilm grown at 360 μEinst m⁻² s⁻¹ and 2.4 mM HCO₃⁻ was limited by the supply of inorganic carbon. Exposure to light above 360 μEinst m⁻² s⁻¹ stimulated gross photosynthesis as well as respiratory processes without affecting net outflux of O₂. Higher concentrations of inorganic carbon, on the other hand, enhanced gross photosynthesis without concurrent increase in respiratory rate, resulting in an increased outflux of O₂. High concentrations of O₂ in the liquid medium decreased the net outflux of O₂ with little effect on the gross photosynthesis. The effects of inorganic carbon and O₂ on the metabolic activities of the biofilm were consistent with the presence of photorespiratory activity.     

The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.