Observer design for differential-algebraic model of an aerobic culture of a recombinant yeast

The mathematical model of an aerobic culture of recombinant yeast presented in work by Zhang et al. (1997) is given by a differential-algebraic system. The classical nonlinear observer algorithms are generally based on ordinary differential equations. In this paper, first we extend the nonlinear observer synthesis to differential-algebraic dynamical systems. Next, we apply this observer theory to the mathematical model proposed in Zhang et al. (1997). More precisely, based on the total cell concentration and the recombinant protein concentration, the observer gives the online estimation of the glucose, the ethanol, the plasmid-bearing cell concentration and a parameter that represents the probability of plasmid loss of plasmid-bearing cells. Numerical simulations are given to show the good performances of the designed observer.Symbols C ₁ activity of pacing enzyme pool for glucose fermentation (dimensionless) - C ₂ activity of pacing enzyme pool for glucose oxidation (dimensionless) - C ₃ activity of pacing enzyme pool for ethanol oxidation (dimensionless) - E ethanol concentration (g/l) - G glucose concentration (g/l) - k _a regulation constant for (g glucose/g cell h^–1) - k _b regulation constant for (dimensionless) - k _c regulation constant for (g glucose/g cell h^–1) - k _d regulation constant for (dimensionless) - K _m1 saturation constant for glucose fermentation (g/l) - K _m2 saturation constant for glucose oxidation (g/l) - K _m3 saturation constant for ethanol oxidation (g/l) - L ( t) time lag function (dimensionless) - p probability of plasmid loss of plasmid-bearing cells (dimensionless) - P recombinant protein concentration (mg/g cell) - q _G total glucose flux culture time (g glucose/g cell h) - t culture time (h) - t _lag lag time (h) - X total cell concentration (g/l) - X ⁺ plasmid-bearing cell concentration (g/l) - Y ^F _{X / G} cell yield for glucose fermentation pathway (g cell/g glucose) - Y ^O _{X / G} cell yield for glucose oxidation pathway (g cell/g glucose) - Y _{X / E} cell yield for ethanol oxidation pathway (g cell/g ethanol) - Y _{E / X} ethanol yield for fermentation pathway based on cell mass (g ethanol·g cell) - ₂ glucoamylase yield for glucose oxidation (units/g cell) - ₃ glucoamylase yield for ethanol oxidation (units/g cell) - µ₁ specific growth rate for glucose fermentation (h^–1) - µ₂ specific growth rate for glucose oxidation (h^–1) - µ₃ specific growth rate for ethanol oxidation (h^–1) - µ_1max maximum specific growth rate for glucose fermentation (h^–1) - µ_2max maximum specific growth rate for glucose oxidation (h^–1) - µ_3max maximum specific growth rate for ethanol oxidation (h^–1)     

Ammonia and O2 uptake in relation to proton translocation in cells of Nitrosomonas europaea

The uptake of ammonia and O₂ by washed cells of Nitrosomonas has been followed simultaneously and continuously using electrode techniques. The stoichiometry of NH ₄ ⁺ oxidation, O₂ uptake and NO ₂ ^- production was 1 : 1.5 : 1.0 and for NH₂OH oxidation a ratio of 1 for O₂ : NO ₂ ^- . A variety of inhibitors of electron transport and metals as well as uncouplers restricted ammonia uptake more markedly than O₂ utilization. There is good evidence for the involvement of copper in the NH ₄ ⁺ uptake process.A quinacrine fluorescence technique has been used to study the proton extrusion by washed cells on adding NH₄Cl and NH₂OH respectively as substrates. The uptake of NH ₄ ⁺ was followed by the extrusion of H⁺ and this process was depressed by those inhibitors which were also effective in the electrode experiments. A requirement for copper is also established for the translocation of protons into the medium, resulting from the uptake of NH ₄ ⁺ by cells.Abbreviations mCCCP carbonyl cyanide m-chlorophenylhydrazone - DBP 2,4 dibromophenol - DCCD N-N-dicyclohexylcarbodimide - DIECA Sodium diethyldithiocarbamate - DNP 2,4 dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - NBD chloride 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - N-serve 2-chloro-6-trichloromethyl-pyridine - PCP pentachlorophenol - 2-TMP 2-trichloromethyl-pyridine - TPB tetraphenylboron - TTFA 1-[thenoyl-(2)]-3,3,3-trifluoracetone - KSCN Potassium thiocyanate     

Embryonic nutrition,growth and energetics inZoarces viviparus L. as indication of a maternal-fetal trophic relationship

Summary The embryos ofZoarces viviparus (L.) show linear growth during their intraovarian development. In early gestation before hatching, the embryos take up very small amounts of low molecular tracer compounds such as glucose, glycine or taurine. Later in gestation (two months after hatching), the embryos accumulate substantial amounts of the tracer compounds. The uptake rates of the tracer compounds in vitro are correlated with ambient concentrations of unlabelled compounds within the natural concentration range of the ovarian fluid. The highest uptake rates are found for glucose and the lowest for taurine. Release of¹⁴CO₂ and dissolved organic carbon (DO¹⁴C) from assimilated tracers in the embryos is low. Oxygen uptake and body weight of the embryos appear to be linearly correlated, and the average oxygen uptake is 4.20 (SD 0.73) mol O₂ g^–1 h^–1 WW at 11°C. The contribution of glucose respiration to total aerobic respiration is 13.9%. A growth to respiration ratio of 0.91 indicates a relatively high efficiency for converting food to growth.Symbols and abbreviations DW dry weight - WW wet weight - DO ¹⁴ C dissolved organic carbon (¹⁴C-labelled) - t _1/2 half life time - turnover time (replacement time)     

Ethanol production with Zymomonas mobilis with synthetic medium in batch operation

Ethanol was produced with Zymomonas mobilis Z6 (ATCC 29191), in batch culture with synthetic medium on glucose as substrate and in the presence of aspartate. The concentrations of glucose, phosphate, ammonium, ethanol and dissolved O₂ and CO₂ in the medium and O₂ and CO₂ in the outlet gas as well as the cell mass by culture fluorescence were measured on-line. Cell mass, glucose and aspartate concentrations were measured off-line. In the presence of a sufficient amount of aspartate, the ethanol inhibition effect can be reduced considerably. However, the improvement with yeast extract is more incisive. The relationship between the intensity of culture fluorescence and cell mass concentration is linear, if sufficient aspartate is present.List of Symbols ASP kg/m³ aspartate concentration - CTR kg/(m³ · h) CO₂ transfer rate - N, NH₄ kg/m³ nitrogen concentration from NH ₄ ⁺ - P kg/m³ product (ethanol) concentration - p% product (ethanol) yield - PO₄ kg/m³ phosphate concentration - Q _E kg/(kg · h) specific ethanol production rate - kg/(kg · h) specific nitrogen uptake rate from NH ₄ ⁺ - Q _P kg/(kg · h) specific phosphate uptake rate - Q _s kg/(kg · h) specific substrate (glucose) uptake rate - S kg/m³ glucose concentration - S _O kg/m³ initial glucose concentration - Y _x/s kg/kg yield coefficient - h^–1 specific growth rate     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Development of a structured model for biopolymer synthesis in the fungus Aureobasidium pullulans

Previous modelling of the pullulan fermentation is discussed and found to lack any mechanistic basis. It is concluded that predictive ability can only be conferred by a structured model with at least two compartments, based upon the best available knowledge of the physiology of the microorganism. Such a model is constructed and compared with experimental data.List of Symbols A (gdm^–3)(g/l) Ammonium ion concentration - B (gdm^–3)(g/l) Concentration of balanced growth compartment of biomass - G (gdm^–3)(g/l) Glucose concentration - k _A (gdm^–3)(g/l) Saturation constant for ammonium - k _G (gdm^–3)(g/l) Saturation constant for glucose - k _S (gdm^–3)(g/l) Saturation constant for sucrose - P (gdm^–3)(g/l) Pullulan concentration - Q Quality of biomass=U/(U+B) - r _G (gdm^–1h^–1)(g/l/h) Rate of removal of glucose from broth - r _GB (gdm^–3h^–1)(g/l/h) Rate of incorporation of glucose into balanced compartment - r _GB (gdm^–3h^–1)(g/l/h) Rate of utilisation of glucose for energy production and cell maintenance - r _GP (gdm^–3h^–1)(g/l/h) Rate of conversion of glucose to pullulan - r _GU (gdm^–3h^–1)(g/l/h) Rate of incorporation of glucose into unbalanced compartment - r _s (gdm^–3h^–1)(g/l/h) Rate of conversion of sucrose to glucose - S (gdm^–3)(g/l) Concentration of sucrose - U (gdm^–3)(g/l) Concentration of unbalanced growth compartment of biomass - X (gdm^–3)(g/l) Biomass concentration - Y _G/A Grams of glucose consumed per gram of ammonium consumed - Y _G/B Grams of glucose consumed per gram of balanced biomass produced - Y _G/U Grams of glucose consumed per gram of unbalanced biomass produced - Y _G/P Grams of glucose consumed per gram of pullulan produced - Rate constant for conversion of sucrose to glucose - Rate constant for uptake of glucose by the cells - Model parameter governing inhibition of sucrose conversion and glucose utilisation - Model parameter denoting fraction of glucose uptake devoted to cell maintenance and energy production - Model parameter governing apportionment of glucose between pseudo-growth and pullulan production This work was funded by the National Engineering Laboratory (NEL) through the Bioreactor Design Club. The authors would like to express their gratitude to the NEL for this generous support.     

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

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

Plasma glucose kinetics and tissue uptake in brown trout in vivo: effect of an intravascular glucose load

The object of the present study was to elucidate whether a glucose load modifies glucose uptake by tissues in brown trout in vivo. By the use of 2-[1,2-³H]-deoxyglucose, plasma glucose disappearance rate and tissue glucose uptake were measured after an intraaortic glucose load of 500 mg·kg^-1 (glucose load group) and under normoglycemic conditions (control). We also attempted to determine whether fasting modifies the glucose load disposal (fasted glucose load group). The procedure used to calculate 2-deoxyglucose uptake by tissues was evaluated, and the levels of 2-deoxyglucose uptake were compared with those of 2-deoxyglucose phosphorylation. Uptake and phosphorylation rates were similar in all tissues, except in brain and heart. In all the groups glucose uptake rates were highest in spleen, kidney, brain and gills, and lowest in red muscle, heart and white muscle. However, white muscle was the main site of glucose uptake on a whole tissue basis. The glucose load led to strong, long-lasting hyperglycemia, in spite of the increases observed in plasma insulin levels and in glucose uptake rate by the whole body (control: 4.9 mol·min^-1·kg^-1; glucose load group: 6.5 mol·min^-1·kg^-1). This higher rate was due to the higher glucose uptake only in white and red muscles (four- and threefold, respectively). Fasting halved the uptake of glucose by both red and white muscles in the load condition. In consequence the use of exogenous glucose decreased with fasting (fasted glucose load group: 5.1 mol·min^-1·kg^-1), causing still longer hyperglycemia.Abbreviations bw body weight - 2DG 2-[1,2-³H]-deoxyglucose - 2DG-P 2-[1,2-³H]-deoxyglucose phosphate - dpm disintegrations per min - FGL fasted glucose load group - GL glucose load group - G-6-Pase glucose-6-phosphatase - LG L-[1-¹⁴C]-glucose - MS-222 3-aminobenzoic acid ethyl ester methanesulphonate salt     

Sucrose uptake in isolated phloem of celery is a single saturable transport system

The uptake of different sugars was studied in segments of isolated phloem from petioles of celery (Apium graveolens L.) in order to determine the kinetics and specificity of phloem loading in this highly uniform conductive tissue. The uptake kinetics of sucrose and the sugar alcohol, mannitol, which are both phloem-translocated, indicated presence of a single saturable system, while uptake of non-phloem sugars (glucose and 3-O-methylglucose) exhibited biphasic kinetics with lower uptake rates than those for sucrose and mannitol. The presence of unlabeled mannitol, 3-O-methylglucose and maltose in the incubation solution did not cause inhibition of labeled-sucrose uptake, indicating high carrier specificity and lack of sucrose hydrolysis in vivo. The pH optimum for sucrose uptake was 5–6. Furthermore, a rapid and transient alkalinization of the external media by sucrose indicated a sugar/H⁺-cotransport mechanism. Dual-labeling experiments showed that sucrose influx continued at a constant rate (V _max=15 mol·h^-1·(g FW)^-1), whereas sucrose efflux was low and insensitive to external concentration. Therefore, the saturable uptake kinetics for sucrose did not appear to be the result of an equilibrium between rates of sucrose influx and efflux.Abbreviations 3-OMG 3-O-methylglucose - PCMBS p-chloromercuribenzene sulfonate - SE-CC sieve element-companion cell - VB vascular bundle     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

Inhibition by nisin of K+ uptake in a sensitivePediococcus sp. strain

Summary A nisin-sensitive strain ofPediococcus sp possessed an uptake system for K⁺ which was apparently dependent on metabolic energy and ATPase activity. K⁺ uptake rate was dependent on the glucose and K⁺ concentrations and showed approximately Michaelis-Menten kinetics with respect to both of these variables with K_t values of 1.2 mM and 599 μM respectively. The presence of nisin inhibited K⁺ uptake with the percentage inhibition proportional to the nisin activity,. Total inhibition occurred at between 4.5 and 5.0 IU ml⁻¹ and the MIC was approximately 0.6 IU ml⁻¹.     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

Cyclic AMP regulation of glucose transport in germinating Pilobolus longipes spores

Pilobolus longipes spores were activated by either glucose or 6-deoxyglucose. Glucose-induced spore activation was previously shown to follow an increase in intracellular cyclic AMP. Concurrent with glucose-induced spore activation, were shifts in 6-deoxyglucose transport kinetics towards higher V _max and K _m values. Cyclic AMP derivatives also caused spore activation and similar changes in the kinetic parameters of 6-deoxyglucose transport. The time course of activation was paralleled by changes in transport activity. Inhibition of phosphodiesterase alone did not cause activation or induce changes in transport activity, but in combination with sub-optimal levels of either 6-deoxyglucose or cAMP derivatives, it amplified the germination signals to produce large increases in both spore activation and 6-deoxyglucose transport activity. These results support the conclusion that glucose transport in germinating spores is regulated by cAMP.Abbreviations IBMX 3-isobutyl-1-methylxanthine; monobutyryl cyclic AMP - N⁶ monobutyryladenosine 3:5-cyclic monophosphate - 8-bromo cyclic AMP 8-bromoadenosine 3:5-cyclic monophosphate     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine     

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     

Enhanced mineralization and denitrification as a result of heterogeneous distribution of clover residues in soil

Batch studies were conducted with Mn oxides (birnessite-hausmannite mixture, BHM) and samples of four soil series from the Mid-Atlantic region of the USA to determine effects of reducing organic acids, similar to those found in the rhizosphere, on the SeO₃/SeO₄ distribution. Jackland (Typic Hapludalf), Myersville (Ultic Hapludalf), Christiana (Aeric Paleaquult), and Evesboro (Typic Quartizipsamment) A and B horizon soil samples with and without prior Mn oxide reduction were incubated aerobically for 10 d with 0.1 mmol kg^-1 SeO₃ and 0 or 25 mmol kg^-1 of ascorbic acid, gallic acid, oxalic acid, or citric acid. Selenite was also added to BHM (10 mmol kg^-1) with 0 or 0.1 mmol kg^-1 ascorbic acid. The availability of Se for plant uptake as a result of root-soil interactions was examined using growth chamber studies with barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) seedlings grown in 150-mL cone-shaped containers to maximize root-soil surface interactions and to create rhizosphere soil throughout the root zone. In the BHM system ascorbic acid increased oxidation of SeO₃ to SeO₄ to 33% of added SeO₃. In the presence of ascorbic and gallic acids and Mn oxides, oxidation of SeO₃ to SeO₄ occurred in the B horizons of all the soils and in the A horizons of Jackland and Myersville soils. Removal of Mn oxides decreased the oxidation in some samples. Wheat and barley plants were able to accumulate up to 20 mol Se kg^-1 from the Jackland soil when soluble Se was not measurable. The root-soil interactions in the Jackland soil with barley and wheat provided the plant with Se from insoluble sources. The results also indicate that Mn oxides coming in contact with reducing root exudates have a greater ability to oxidize SeO₃ to SeO₄. Thus, rhizosphere processes play an important role in the availability of Se for plant uptake.Maryland Agricultural Experiment Station Scientific Article A 6381.Maryland Agricultural Experiment Station Scientific Article A 6381.     

Lactate oxidation byTreponema pallidum

Whole cells ofTreponema pallidum consumed O₂ with lactate in a glucose-depleted medium.d(–) Lactate caused marked stimulation of O₂ uptake at a rate similar to that with glucose, whereasl(+) lactate resulted in no increase over the reduced rate observed upon glucose depletion. Lactate oxidation was specific for -hydroxy straight-chain acids of 3,4, and 5 carbons. O₂ uptake during lactate oxidation proceeded independently of pyruvate oxidation and required NAD. The product of lactate oxidation was pyruvate.d(–) Lactate-stimulate O₂ uptake was sensitive to chlorpromazine and resistant to amytal and cyanide. Glucose did not inhibit the oxidation of lactate as shown by the additive effect of both substrates on O₂ uptake. Oxidation of glucose, but not lactate, provided energy necesary for motilibty or maintenance of virulence. A mixture of lactate isomers was formed from glucose with thel(+) isomer concentration remaining constant and thed(–) isomer concentration varying inversely with dissolved O₂ concentration. The function of lactate as an oxidizable substrate is apparently quite distinct from that of glucose.