Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O₂ to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium. Correspondence to: D. Levanon     

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y _glucose ^max and values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h^–1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K⁺ or Mg²⁺)-limited cultures expressed the highest metabolic rates and with the K⁺ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K⁺-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K⁺ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.     

Influence of medium frequency light/dark cycles of equal duration on the photosynthesis and respiration of Chlorella pyrenoidosa

Chlorella pyrenoidosa was grown in three continuous cultures each receiving a different light regime during the light period of a diurnal cycle. Hourly samples taken during the light period were subjected to medium frequency light/dark oscillations of equal duration, ranging from 3 to 240 seconds. The oxygen consumption and production of each sample were measured with an oxygen electrode in a small oxygen chamber. Although the light/dark cycles had little overall influence on photosynthetic activity, the microalgae appeared to adapt to the light regime to which they were subjected. Large differences were found between the maximum chlorophyll-specific production rates (P _infmax ^supB ), the chlorophyll-specific production rates (P^B) and the respiration rates between the cultures and treated subsamples. Respiration rates increased during the light period, whilst P^B either increased, or had a mid light period minimum or maximum. The culture which received an hourly light oscillation during the light period had the highest P _infmax ^supB and lowest respiration rates, and it is suggested that these algae react as in nature, whereas either a sinusoidal or a block light pattern is unnatural. The latter light regime is commonly used in laboratory studies.     

Salt stress and respiration inAspergillus repens

Studies with whole cells and mitochondrial fractions revealed increased respiratory activity inAspergillus repens grown under salt stress conditions. The state 3 and state 4 respiration rates, PO ratios, and Mg²⁺-dependent ATPase were higher in mitochondria of stressed cells than in control cells.A. repens respires via an antimycin A-and cyanide-sensitive pathway. Oligomycin, dicyclohexylcarbodiimide (DCCD) and rotenone inhibited respiration rates less in mitochondria of stressed cells than in controls. Though 2,4-dinitrophenol (DNP), carbonyl cyanide-m-chlorophenylhydrazone (m-Cl-CCP), and carbonyl cyanide-p-trifluoromethylhydrazone (p-F₃-CCP) did not stimulate Mg²⁺-ATPase activity, DNP enhanced the respiration rates, whereasm-Cl-CCP andp-F₃-CCP decreased the respiration rates in either condition; mitochondria of stressed cells exhibited a lower degree of inhibition than controls. Addition of DNP, oligomycin, and DCCD inhibited the basal Mg²⁺-ATPase (ATPase activity without Mg²⁺ addition). Oligomycin inhibited the Mg²⁺-ATPase. DCCD showed less inhibition in mitochondria under stress than did the controls. Levels of some respiratory enzymes were higher in the culture grown under stress than in the controls.     

The effect of dynamic light regimes on Chlorella II. Minimum quantum requirement and photosynthesis-irradiance parameters

Comparisons were made of photosynthesis in three light limited cyclostat cultures (LD = 8:16, dilution rate 0.7 d^–1) of Chlorella pyrenoidosa, differing only in the dynamics of irradiance supply: as a constant rate, i.e. a block culture; as a sine function of the light period, i.e. a sinusoidal culture; as an 8 h sine function superimposed by an 1 h sine function, i.e. an oscillating culture. The sinusoidal culture had a constant minimum quantum requirement for oxygen evolution (QR) of 10.8 over the photoperiod. The OR of the oscillating culture increased from 24 to 37 during the photoperiod. From changes in and P _max we suggest that: (1) photosynthetic units (PSU) of the block and sinusoidal sulture increased in number; (2) increasingly fewer chlorophyll molecules participated in oxygenic photosynthesis with a decreasing turnover time of the PSU's during an oscillating photoperiod. Values of I _k decreased slightly in the block culture, increased slightly in the sinusoidal culture and showed a twofold increase in the oscillating culture. From the ratio of in situ oxygen production (qO₂) and P _max we infer a balanced equilibrium between photosystem activity and electron transport capacity for the block and sinusoidal culture. We hypothesize that the qO₂ values of the oscillating culture underestimated true oxygen production rates due to a nonlinear response at peak light intensities. The results show that a dynamical photoperiod provoked significantly different photosynthetic responses, even though the overall growth rate was unaffected.     

Inhibitory effect of dihydroxyacetone onGluconobacter oxydans: Kinetic aspects and expression by mathematical equations

Summary Microbial conversion of glycerol into dihydroxyacetone (DHA) byGluconobacter oxydans was subjected to inhibition by excess substrate. Comparison of cultures containing increasing initial DHA contents (0 to 100 g l^–1) demonstrated that DHA also inhibited this fermentation process. The first effect was on bacterial growth (cellular development stopped when DHA concentration reached 67 gl^–1), and then on oxidation of glycerol (DHA synthesis only occurred when the DHA concentration in the culture medium was lower than 85 g l^–1). Productivity, specific rates and, to a lesser extent, conversion yields decreased as initial concentrations of DHA increased. The changes in the specific parameters according to increasing initial DHA contents were described by general equations. These formulae satisfactorily express the concave aspect of the curves and the reduction in biological activity when the cells were in contact with DHA concentrations of up to 96 g l^–1.Abbreviations X, S, P biomass, substrate, product concentrations - r _x,r _s,r _p rates of growth, consumption and production - ,q _s,q _p specific rates of growth, glycerol consumption and DHA production - Y _x/s, Y_p/s conversion yields of substrate into biomass and product - K _s constant of affinity of cells to the substrate - K _ip product inhibition constant - P _m threshold concentration of DHA in substrate     

Kinetics of yeast growth and enzyme syntheses in a phosphate-limited continuous culture

Summary The effect of a deficiency of inorganic phosphate on the growth rate and on the invertase and phosphatase activities inSaccharomyces carlsbergensis was studied in a chemostat culture using a synthetic medium in which ethanol was the sole carbon source.The kinetic relationship between the growth rate and both the rates of phosphate uptake and the ethanol consumption agreed well with the threshold model but not the multicative model. The invertase activity of the yeast increased as the dilution rate decreased. As the phosphate concentration in the feed was reduced, the enzyme synthesis increased remarkably. Acid phosphatase activity was repressed completely above a critical molecular ratio, 0.015, of monopotassium phosphate to ethanol in the feed medium. As the phosphate concentration in the feed decreased, the maximum specific enzyme activity increased and the corresponding optimum dilution rate decreased. These experimental changes in enzyme synthesis were expressed mathematically using the modified operon models for enzyme regulation in terms of two fractions of limited inorganic phosphate; one which affects growth and the other which is incorporated in excess by the cells.Nomenclature A ethanol concentration in the culture (mM) - a, b, c, d exponents in the operon model - D dilution rate (h^–1) - E enzyme concentration in the culture (enzyme unit l^–1) - K_a, K_b, K_c, K_d, k equilibrium constants used in the operon model, see Toda (1976b) - o operator gene - P inorganic phosphate concentration in the culture (mM) - Pi limited inorganic phosphate concentration in the cells (mmole inorganic phosphate/g dry weight of cell) - Q specific enzyme activity, no units: (E/X)/(E/X)_max - Q_c, Q_d as defined in Eq. 12 - R repressor - r regulator gene - X cell concentration in the culture (dry cell weight l^–1) Greek Letters molecular ratio of inorganic phosphate to ethanol in the feed medium (mole/mole) - specific growth rate (h^–1) - A specific uptake rate of ethanol (mmole/g cell·h) - P specific uptake rate of inorganic phosphate (mmole/g cell·h) Suffix crit critical value - f feed - max maximum - min minimum - t total - 1, 2 number of species Superfix eff effective for cell growth - exc excess - str structural     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Trehalase activity in mycorrhizal and nonmycorrhizal roots of leek and soybean

Root extracts of leek (Allium porrum L.) and soybean (Glycine max L. Merr.) showed trehalase activity which was inhibited by phloridzin and was several times higher than the activity of general -glucosidase. The activity had an acidic optimum. Trehalase activity in extracts of sporocarps and extraradical mycelium of the arbuscular mycorrhizal fungus Glomus mosseae Nicol. & Gerd. (Trappe & Gerd.) was higher than in root extracts and had an optimum at pH 7. Following inoculation with G. mosseae, trehalase activity increased in mycorrhizal roots above the levels observed in nonmycorrhizal roots. Irrespective of fungal colonization, root trehalase activity increased in the presence of Mg²⁺, decreased in the presence of Mn²⁺ and Zn²⁺, and was unaffected by Na₂EDTA.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

16O2/18O2 analysis of oxygen exchange in Dunaliella tertiolecta. Evidence for the inhibition of mitochondrial respiration in the light

A mass spectrometric ¹⁶O₂/¹⁸O₂-isotope technique was used to analyse the rates of gross O₂ evolution, net O₂ evolution and gross O₂ uptake in relation to photon fluence rate by Dunaliella tertiolecta adapted to 0.5, 1.0, 1.5, 2.0 and 2.5 M NaCl at 25°C and pH 7.0.At concentrations of dissolved inorganic carbon saturating for photosynthesis (200 M) gross O₂ evolution and net O₂ evolution increased with increasing salinity as well as with photon fluence rate. Light compensation was also enhanced with increased salinities. Light saturation of net O₂ evolution was reached at about 1000 mol m^-2s^-1 for all salt concentrations tested. Gross O₂ uptake in the light was increased in relation to the NaCl concentration but it was decreased with increasing photon fluence rate for almost all salinities, although an enhanced flow of light generated electrons was simultaneously observed. In addition, a comparison between gross O₂ uptake at 1000 mol photons m^-2s^-1, dark respiration before illumination and immediately after darkening of each experiment showed that gross O₂ uptake in the light paralleled but was lower than mitochondrial O₂ consumption in the dark.From these results it is suggested that O₂ uptake by Dunaliella tertiolecta in the light is mainly influenced by mitochondrial O₂ uptake. Therefore, it appears that the light dependent inhibition of gross O₂ uptake is caused by a reduction in mitochondrial O₂ consumption by light.Abbreviations DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - DHAP dihydroxy-acetonephosphate - DIC dissolved inorganic carbon - DR_a rate of dark respiration immediately after illumination - DR_b rate of dark respiration before illumination - E₀ rate of gross oxygen evolution in the light - NET rate of net oxygen evolution in the light - PFR photon fluence rate - RubP rubulose-1,5-bisphosphate - SHAM salicyl hydroxamic acid - U₀ rate of gross oxygen uptake in the light     

Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na⁺ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO₃. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na⁺-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na⁺-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

     

Involvement of ethylene in the morphological and developmental response of rice to elevated atmospheric CO2 concentrations

We tested the hypothesis that increased carbohydrate flux under elevatedCO₂ regulates accelerated development using rice (Oryzasativa L. cv. Jarrah). Plants were grown either in flooded soil orsolution culture at either 360 or 700 L CO₂L^–1. Total dry mass, shoot elongation rates (SER),tiller appearance rates (TAR) and ethylene release from intact rice seedlingswere measured from 5 to 42 days after planting (DAP). At maturity, shoot andsheath length, tiller number and grain mass were also measured. ElevatedCO₂ had a profound effect on growth, morphology and development andthe effects were more pronounced during the early growth phase. Total aboveground biomass increased at elevated CO₂ and this was accounted for by enhanced tiller number. Grain yield was increased by 56% under elevated CO₂mainly due to increased tiller number and hence panicle number. TAR and SERwereenhanced at elevated CO₂ but SER increased only untill 25 DAP.Elevated CO₂ stimulated a 2-3-fold increase in endogenous andACC-mediated ethylene release but the ACC concentration in the leaves waslittleaffected showing that rates of ACC synthesis matched its oxidation. Inhibitionof ethylene action by 1-aminocyclopropane (1-MCP) had a more pronouncedinhibitory effect on ethylene release in plants that were grown at 700 ascompared to 360 L CO₂ L^–1. Feedingsucrose to intact plants enhanced ethylene synthesis and these results areconsistent with the hypothesis that increased accumulation of sucrose atelevated CO₂ may enhance expression of genes in the ethylenebiosynthetic pathway. We conclude that increase in ethylene release may becentral in promoting accelerated development under elevated CO₂ andthis coincides with the release of auxiliary buds and accelerated rates oftiller appearance hence increased grain yield at elevated CO₂.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

Effect of the cationic polypeptide polylysine on neutral amino acid transport in isolated brain microvessels

Summary The functionality of isolated brain microvessels — used as anin vitro model of the blood-brain barrier — can be influenced by interaction with cationic proteins. The various polylysines (M_r ranging from 0.9 to 180 kDa) tested affected the activity of both the Na⁺-dependent (A) and the Na⁺-independent (L) systems for neutral amino acid transport. Exposure to the 180 kDa polylysine caused a conspicuous inhibition of both transport systems, associated to an increased passive permeability. There was a constant, M_r-dependent, inhibition of the the L-system-mediated uptake of hydrophobic neutral amino acids. The activity of the A-system was enhanced, upon exposure to polymers larger than 22 kDa reaching its peak at 68 kDa and and declining at higher M_r values. The effect which was Na⁺-ions dependent and abolished by phloretine, could be essentially ascribed to an increased affinity of the MeAIB for its carrier (K_m value decreasing from 265 to 169µM in presence of 68 kDa polylysine).