Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

Relative toxicity of nC24 agricultural mineral oil to Tetranychus urticae Koch (Acari: Tetranychidae) and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) and its possible relationship to egg ultrastructure

The relative toxicity (LC₅₀ values based on µg oil/cm²) is evaluated of aqueous n C24 agricultural mineral oil (AMO) emulsions to the egg, six-legged nymph (larva), eight-legged protonymph and adult stages of two-spotted mite ( Tetranychus urticae ) and its predator, Phytoseiulus persimilis , on French bean leaf discs, using a Potter spray tower to apply of the oil. The egg of P. persimilis was the least susceptible stage (LC₅₀ 444.84) and its LC₅₀ was significantly higher than all other stages tested of either P. persimilis or T. urticae . The LC₅₀ for adult female T. urticae (LC₅₀ 63.89) was significantly lower than the larva (LC₅₀ 93.86); however, there was no significant difference in response between the protonymph (LC₅₀ 70.44) and the larva, which were both higher than T. urticae eggs (LC₅₀ 17.55). LC₅₀s for P. persimilis larva (LC₅₀ 43.87), protonymph (LC₅₀ 41.55) and adult female (LC₅₀ 53.34) were similar. Scanning electron microscopy showed that the egg surface of T. urticae is usually well covered with fine silk that may trap more oil and increase AMO efficacy. Other possible differences in AMO efficacy between T. urticae and P. persimilis may be due to differences in egg size, egg incubation period, egg surface structure and the presence of vulnerable respiratory cones in T. urticae eggs. Dose of 0.2–0.3% (w/w) is considered to be the most appropriate for n C24 AMOs use against T. urticae in combination with P. persimilis in integrated pest management programs.     

The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Water Relations of Glucose-catabolizing Enzymes in Pseudomonas fluorescens

Examination of the catabolism of glucose via the Entner-Doudoroff pathway by standard enzyme assays showed that the activity of glucose-6-phosphate dehydrogenase, glucokinase and 2-ketoglu-conokinase plus phosphoketogluconate reductase was completely inhibited at a _w values less than 0.965, 0.98 and 0.96 respectively when NaCl was used to adjust the a _w. The other glucose-catabolizing enzymes were inhibited to a lesser degree. When sucrose was used to control a _w, glucokinase and glucose-6-phosphate dehydrogenase were inhibited at 0.92 a _w but the other enzymes remained active below 0.86 a _w. Enzymes were relatively active at reduced a _w when adjusted with glycerol and most remained active even at 0.80 a _w. When a _w was controlled by potassium glutamate, the activity of glucokinase and glucose-6-phosphate dehydrogenase was markedly less inhibited than by NaCl at similar a _w. Possible reasons for the variation in activity by glucose-catabolizing enzymes in response to a _w controlled by various solutes could be location of the enzyme in the cell, ability of the solute to penetrate the cell and ability to withstand high salt and sucrose concentrations. When the a _w of the growth medium was reduced to 0.98 by glycerol, NaCl and polyethylene glycol 400, levels of glucokinase were significantly reduced while higher levels of glucose dehydrogenase and gluconate dehydrogenase were induced. This suggests that reduction in a _w could regulate the routes of catabolism in the Entner-Doudoroff pathway. When sucrose was used to control a _w of the growth medium high levels of most enzymes were induced, suggesting catabolism of the sucrose by the organism.     

Redox modulation of homomeric rho1 GABA receptors

The activity of many receptors and ion channels in the nervous system can be regulated by redox-dependent mechanisms. Native and recombinant GABA_A receptors are modulated by endogenous and pharmacological redox agents. However, the sensitivity of GABA_C receptors to redox modulation has not been demonstrated. We studied the actions of different reducing and oxidizing agents on human homomeric GABAρ₁ receptors expressed in Xenopus laevis oocytes. The reducing agents dithiothreitol (2 mM) and N -acetyl- l -cysteine (1 mM) potentiated GABA-evoked Cl⁻ currents recorded by two-electrode voltage-clamp, while the oxidants 5-5'-dithiobis-2-nitrobenzoic acid (500 μM) and oxidized dithiothreitol (2 mM) caused inhibition. The endogenous antioxidant glutathione (5 mM) also enhanced GABAρ₁ receptor-mediated currents while its oxidized form GSSG (3 mM) had inhibitory effects. All the effects were rapid and easily reversible. Redox modulation of GABAρ₁ receptors was strongly dependent on the GABA concentration; dose–response curves for GABA were shifted to the left in the presence of reducing agents, whereas oxidizing agents produced the opposite effect, without changes in the maximal response to GABA and in the Hill coefficient. Our results demonstrate that, similarly to GABA_A receptors and other members of the cys-loop receptor superfamily, GABA_C receptors are subjected to redox modulation.     

Inhibitory effect of sodium bicarbonate on the motility of sperm of Japanese eel

Sperm motility of Japanese eel Anguilla japonica was depressed from 59.1 to 1.1% when NaHCO₃ concentration increased from 0 to 150 mM. In 25 mM NaHCO₃, when pH of the medium was 6.2, 7.2, 8.2, 9.2 and 10.2, relative sperm motility was 0, 0, 55.8, 93.7 and 136.6%, respectively to that of the control (0 mM NaHCO₃). The remarkable effect in acid or neutral condition indicates that free-CO₂ (liberated CO₂ and H₂CO₃) is a key factor for the motility inhibiting effect of NaHCO₃.     

Mechanism of the inhibition of phloem loading by sodium sulfite: Effect of the pollutant on the transmembrane potential difference

Sodium sulfite (Na₂SO₃) decreased uptake from 1 m M sucrose by the parenchyma and by the veins of leaves of broadbean ( Vicia faba L. cv. Aguadulce). The decrease depended on the concentration of the pollutant and the duration of pretreatment. The inhibition was non-competitive. Sulfite affected the transmembrane potential difference (PD) of the leaf tissues. The short-term response obeyed an 'all or nothing' law. At 0.1 m M and above, sulfite led to a quick depolarization of one-third of the initial potential after a lag phase of about 5 min; for concentrations lower than 0.1 m M , sulfite did not affect the potential. By contrast, the long-term effect of Na₂SO₃ on the transmembrane PD strongly depended on its concentration. After 2–12 h of pretreatmemt there was no effect at 10 μ M , a weak effect at 0.1 m M , and then increasing depolarization as the pollutant concentration increased. The inhibitory effect of Na₂SO₃ on sucrose uptake is thus, at least partly, due to its effect on a component of the proton-motive force. ΔΨ. However, the lack of correlation noticed with 0.1 m M Na₂SO₃ between the effect on sucrose uptake and the long-term effect on transmembrane PD suggests numerous sites of sulfite action.     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Maximal biomass production can occur in corn (Zea mays) in the absence of NO3 accumulation in either leaves or roots

In order to investigate effects of limited NO₃ availability in corn ( Zea mays L. cv. Brulouis) 17-day-old plants were grown for a further 25 days on sand in a growth chamber. The plants received frequent irrigation with a complete nutrient solution containing 0.2, 0.6, 1.5 or 3.0 mM NO₃. With 0.2 mM NO; nitrate levels in both roots and leaves diminished rapidly and were almost zero after 10 days treatment. Concurrently, as signs of nitrogen deficiency appeared, shoot growth was restricted, whereas root growth was enhanced. In addition, the concentration of reduced nitrogen and malate in the leaves declined, and in vitro nitrate reductase activity (NRA. EC 1.6.6.1), soluble protein and chlorophyll levels of leaf tissue were depressed and starch concentration was enhanced. With 0.6 mM NO₃ in the nutrient solution, the decrease in NO₃ levels in the tissues and the increase in root development were similar to those observed with 0.2 mM NO₃. However, shoot growth, reduced nitrogen concentration in leaves, and the above-mentioned biochemical characteristics were almost identical to those obtained at 1.5 and 3.0 mM NO₃. This indicates that when supplied with 0.6 mM NO₃, corn plants were able to absorb sufficient NO3 to support maximal biomass production without appreciable NO₃ accumulation in roots or shoot. It is, thus, suggested that the plants responded to low NO₃, availability in medium by enhancing root growth and by maximizing NO₃ reduction relative to NO₃ accumulation.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

The effects of increased atmospheric carbon dioxide and temperature on carbon partitioning, source-sink relations and respiration

Abstract. Herbaceous C₃ plants grown in elevated CO₂ show increases in carbon assimilation and carbohydrate accumulation (particularly starch) within source leaves. Although changes in the partitioning of biomass between root and shoot occur, the proportion of this extra assimilate made available for sink growth is not known. Root:shoot ratios tend to increase for CO₂-enriched herbaceous plants and decrease for CO₂-enriched trees. Root:shoot ratios for cereals tend to remain constant. In contrast, elevated temperatures decrease carbohydrate accumulation within source and sink regions of a plant and decrease root:shoot ratios. Allometric analysis of at least two species showing changes in root: shoot ratios due to elevated CO₂ show no alteration in the whole-plant partitioning of biomass. Little information is available for interactions between temperature and CO₂. Cold-adapted plants show little response to elevated levels of CO₂, with some species showing a decline in biomass accumulation. In general though, increasing temperature will increase sucrose synthesis, transport and utilization for CO₂-enriched plants and decrease carbohydrate accumulation within the leaf. Literature reports are discussed in relation to the hypothesis that sucrose is a major factor in the control of plant carbon partitioning. A model is presented in support.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.     

Electrophysiological response of honey bee labial palp contact chemoreceptors to sugars and electrolytes

ABSTRACT. The sensilla chaetica on segments II, III and IV of honey-bee labial palps were investigated electrophysiologically. The responses (spikes/s) correlated with the log of the concentrations of sucrose, glucose, fructose, NaCl, KCl and LiCl, but not with CaCl₂ or MgCl₂, which gave inconsistent responses. The firing rates were higher and thresholds lower to the sugars than to the electrolytes. The sensitivity of the segments fell in the order: III > II > IV for most of the stimulants, which elicited responses in the order: sucrose > glucose = fructose' KCl > LiCl > NaCl. The sensilla adapted logarithmically with time. No synergism of response was noted when mixed-sugar solutions were applied, but inhibition of response was seen when glucose–sucrose, fructose–sucrose, and glucose–fructose–sucrose mixtures were applied. None of the sensilla tested responded to water.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.