Updated model of the batch acetone-butanol fermentation

Summary The recent models of the Acetone-Butanol fermentation did not adequately describe the culture inhibition by the accumulating metabolites and were unable to simulate the acidogenic culture dynamics at elevated pH levels. The present updated modification of the model features a generalised inhibition term and a pH dependent terms for intracellular conversion of undissociated acids into solvent products. The culture dynamics predictions by the developed model compared well with experimental results from an unconventional acidogenic fermentation ofC. acetobutylicum.Nomenclature A acetone concentration in the fermentation broth, [g/L] - AA total concentration of dissociated and undissociated acetic acid, [g/L] - AA _undiss concentration of undissociated acetic acid, [g/L] - APS Absolute Parameter Sensitivity - AT acetoin concentration in the fermentation broth, [g/L] - B butanol concentration in the fermentation broth, [g/L] - BA total concentration of dissociated and undissociated butyric acid, [g/L] - BA _undiss concentration of undissociated butyric acid, [g/L] - E ethanol concentration in the fermentation broth, [g/L] - f(T) inhibition function as defined in Equation (2) - k ₁ constant in Equation (4), [g substrate/g biomass] - k ₂ constant in Equation (4), [g substrate/(g biomass.h)] - k ₁ constant in Equation (5), [g substrate/(g biomass] - k ₂ constant in Equation (5), [g substrate/(g biomass.h)] - k ₃ constant in Equation (6), [g butyric acid/g substrate] - k ₄ constant in Equation (6), [g butyric acid/(g biomass.h)] - k ₅ constant in Equation (7), [g butanol/g substrate] - k ₆ constant in Equation (8), [g acetic acid/g substrate] - k ₇ constant in Equation (8), [g acetic acid/(g biomass.h)] - k ₈ constant in Equation (9), [g acetone/g substrate] - k ₉ constant in Equation (10), [g ethanol/g substrate] - k ₁₀ constant in Equation (11), [g acetoin/g substrate] - k ₁₁ constant in Equation (12), [g lactic acid/g substrate] - K _I Inhibition constant, [g inhibitory products/L] - ke maintenance energy requirement for the cell, [g substrate/(g biomass.h)] - K _AA acetic acid saturation constant, [g acetic acid/L] - K _BA butyric acid saturation constant, [g butyric acid/L] - K _S Monod's saturation constant, [g substrate/L] - LA lactic acid concentration in the fermentation broth, [g/L] - m _i ,n _i constants in Equation (14) - n empirical constant, dependent on degree of inhibition. - P concentration of inhibitory products (B+BA+AA), [g/L] - P _max maximum value of product concentration to inhibit the fermentation, [g/L] - pK_a equilibrium constant - r _A rate of acetone production, [g acetone/L.h] - r _AA rate of acetic acid production, [g acetic acid/L.h] - r _AT rate of acetoin production, [g acetoin/L.h] - r _B rate of butanol production, [g butanol/L.h] - r _BA rate of butyric acid production, [g butyric acid/L.h] - r _E rate of ethanol production, [g ethanol/L.h] - RPS Relative Parameter Sensitivity - r _LA rate of lactic acid production, [g lactic acid/L.h] - r _S dS/dt=total substrate consumption rate, [g substrate/L.h] - r _S substrate utilization rate, [g substrate/L.h] - S substrate concentration in the fermentation broth, [g substrate/L] - S ₀ initial substrate concentration, [substrate/L] - t time, [h] - X biomass concentration, [g/L] - Y _X yield of biomass with respect to substrate, [g biomass/g substrate] - Y _{P i} yield of metabolic product with respect to substrate, [g product/g substrate] Derivatives dX/dt rate of biomass production, [g biomass/L.h] - dP _i /dt rate of product formation, [g product/L.h] Greek letters specific growth rate of the culture, [h^–1] - I specific growth rate of the culture in the presence of the inhibitory products, [h^–1] - µ_max maximum specific growth rate of the culture, [h^–1]     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Integral kinetics of the cleavage of maltose catalysed by α-glucosidase fromSaccharomyces carlsbergensis

Summary A new, sensitive and continuous assay for -glucosidase is described exploiting the different angles of rotation for the substrate maltose and the product glucose. Kinetic experiments revealed a very pronounced product inhibition of -glucosidase fromSaccharomyces carlsbergensis with a K_i of 4.85·10^–3 M for glucose.The K_M of maltose was found to be 37.8·10^–3 M. Taking these values, an integral kinetic curve for the enzymatic hydrolysis of maltose was calculated, which is shown to fit the experimental data.Symbols used k₁ (min^–1) pseudo first-order rate constant (for enzymatic cleavage) - k₂ (min^–1) rate constant (for mutarotation reaction) - I, P (mol/1) inhibitor (product) concentration - k_i (mmol/1) inhibitor constant - K_M (mmol/l) Michaelis constant - [M] ₅₈₉ ³⁰ (degree/m · l/mol) molecular rotation at 30°C and 589 nm - s (mmol/l) substrate concentration - R (mmol/mg · min) reaction rate - V_max (mmol/mg · min) maximal rate - U (mol/min) activity unit (here at 30°C and pH=6.8) Indices O initial value - max maximal value     

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K⁺ concentrations ([K⁺] _o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V _i) were the same for different [K⁺] _o . For the range ofV _i examined, K⁺ efflux occurs principally through the K⁺ inward rectifier channels. When external K⁺ is removedV _i remains constant for 2 to 3 hr because of the high membrane conductance to Cl^–, but K⁺ efflux drops by about one order of magnitude.External Ba²⁺ in the presence or absence of external K⁺ produces an inhibition of K⁺ efflux described by a relation of the formu=(u₁/(1+C)[Ba²⁺] _o ))+u ₂, whereu is the uninhibited fraction of K⁺ efflux;u ₁, u₂ andC are constants; andu ₁+u₂=1.C depends both on [K⁺] _o andV _i. When [K⁺] _o 75mm, increasing [K⁺] _o at constantV _i reduces Ba²⁺ sensitivity. For constantV _i–30 mV, Ba²⁺ sensitivity is less when [K⁺] _o =0 than when [K⁺] _o 75mm. When [K⁺] _o =0, Ba²⁺ sensitivity decreases asV _i is made more positive. The dependence of the Ba²⁺ sensitivity onV _i at constant [K⁺] _o is greater when [K⁺] _o =0 than when [K⁺] _o 75mm.Both the activation of K⁺ efflux by external K⁺ and the Ba²⁺ inhibition of K⁺ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K⁺, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba²⁺ occupies either site, K⁺ efflux is further reduced. The reduction of Ba²⁺-sensitivity by increasing [K⁺] _o at high [K⁺] _o is attributable to the displacement of Ba²⁺ from the control sites by K⁺. The increased Ba²⁺ sensitivity produced by going from [K⁺] _o =0 to [K⁺] _o >-75mm whenV _i–30 mV is attributable to states in which Ba²⁺ occupies one site and K⁺ the other when [K⁺] _o 0. The smallerV _i dependence of the Ba²⁺ sensitivity when [K⁺] _o 75mm compared to [K⁺] _o =0 is attributable to the necessity that Ba²⁺ displace K⁺ at the control sites when [K⁺] _o is high but not when [K⁺] _o =0.     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Product formation during batch fermentation with recombinant Escherichia coli containing a runaway plasmid

The product formation during batch fermentation with recombinant E. coli containing a runaway replication plasmid has been examined. Theoretical modelling is combined with experimental work to study the effect of operating conditions. In particular the influence of induction profile has been investigated. High sensitivity to operating conditions is observed, and both model and experimental data illustrate the presence of very narrow limits for an optimal induction profile.List of Symbols f _i function for allocation of energy to the i'th reaction in the one substrate model - g _i function for allocation of energy to the i'th reaction in the two substrate model - h function for inhibition by plasmid material - K _i (h^–1) kinetic rate constant for the i'th reaction - k _i (g/l) saturation constants - K _p (g P/g biomass) saturation constant for recombinant protein synthesis - K _s (g/l) inhibition constant of glucose on acetate metabolism - K _p,i (g P/g biomass) inhibition constant of plasmid material on cellular activity - p (g/l) extracellular acetic acid concentration - r _i (h^–1) specific rate of i'th reaction - s (g/l) extracellular glucose concentration - X _i (g i/g biomass) intracellular concentration of the i'th component - _ij stoichiometric coefficients for the i'th metabolic product in the j'th reaction - _ij stochiometric coefficients for the i'th component in the biotic phase in the j'th reaction - _i relative allocation of energy to the i'th reaction with growth on acetate compared with growth on glucose     

Procaine isothiocyanate: An irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels

[³H]Batrachotoxinin-A benzoate ([³H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [³H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative ([³H]PRIT) have been prepared. PRIT inhibits the binding of [³H]BTX-B in a noncompetitive, irreversible manner (apparent K_i=13 M) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (K_i=40 M). The dissociation rate of [³H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [³H]BTX-B is achieved in the presence of 0.98 M PRIT. [³H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Binding characteristics of [3H]flunitrazepam in pentobarbital-withdrawal rats

The effects of chronic pentobarbital (PB) treatment on the binding characteristics of [³H]flunitrazepam (FLU) in rat brain were examined. Saline or sodium PB (500 g/10l/hr) was infused into the lateral cerebral ventricles of rats for 6 days using osmotic pumps. Immediately before withdrawal, there were no significant differences in [³H]FLU binding constants (K_D and B_max) between saline and PB groups. However, 24 hr withdrawal caused an increase in B_max with no changes in K_D. The enhancement of [³H]FLU binding by in vitro addition of chloride ions and PB was not affected after the PB infusion. The PB enhancement of [³H]FLU binding was inhibited by the convulsant, picrotoxicin. PB withdrawal did not cause significant differences in the binding constants of [³H]Ro 15-1788, a benzodiazepine (BZ) antagonist, between the saline and PB groups. Pretreatment of membranes with 0.02 mM of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, caused decreases in both K_D and B_max in FLU binding in PB-withdrawal membrane, but not in the saline-treated membrane. The enhancement of [³H]FLU binding by chloride ions and PB was not affected by the CHAPS treatment. These results suggest that the change in BZ receptors induced by PB withdrawal is functionally linked to the GABA-BZ-barbiturate receptor complex and that PB withdrawal induces some conformational changes in BZ receptors.     

Biotransformation of cephalosporin C to 7-aminocephalosporanic acid with coimmobilized biocatalyst in a batch bioreactor

Biotransformation of cephalosporin C (CPS-C) to 7-aminocephalosporanic acid (7-ACA) was carried out with coimmobilized permeabilized cells of Trigonopsis variabilis and Pseudomonas species entrapped in Ca-pectate gel beads. Good aeration and stirring during the process was assured. The analysis of this complicated biochemical process in a heterogeneous system was based on the identification of individual effects (internal diffusion, reaction) running simultaneously. A spectrophotometric method was proposed for the determination of 7-(-ketoadipyl amido) cephalosporanic acid (CO-GL-7-ACA) and 7-ACA. The reaction-diffusion model containing dimensionless partial differential equations was solved by using the orthogonal collocation method. A good agreement between experimental values and values predicted by the mathematical model was obtained. Numerical simulations were performed on the basis of following the two assumptions:- several times higher activity of both cells,- hydrogen peroxide was continuously supplied in the bioreactor.List of Symbols A m² surface of the bead - c _i mol/dm³ concentration of component in the bead and/or in the solution - c _i0 mol/dm³ initial concentration of component in the solution - c _l0 mol/dm³ initial concentration of CPS-C in the solution - C _jl orthogonal collocation weights of the first derivation - D _ei m²/s effective diffusion coefficient of the components - D _jl orthogonal collocation weights of the second derivation - k ₅ dm³/(mol · s) kinetic parameter of non-enzyme reaction - K _inh mol/dm³ inhibition parameter for the first enzyme reaction - K _i dimensionless Michaelis constant for the first and second enzyme reaction, defined in Eq. (7) - K _l dimensionless inhibition parameter for the first enzyme reaction, defined in Eq. (7) - K _mi mol/dm³ Michaelis constant for the first and second enzyme reaction - n number of beads - P( _i ) symbol of dimensionless reaction rate, defined in Eq. (13) - r m radial coordinate inside the bead - R m radius of the bead - R(c _i ) mol/(dm³ · s) symbol for reaction rate, defined in Eq. (6) - t s time - V _max mol/(dm³ · s) max. reaction rate for the first and second enzyme reaction - V _L dm³ volume of solution excluding the space occupied by beads - voidage in batch bioreactor - _P porosity of the bead - i dimensionless effective diffusion coefficient of the components, defined in Eq. (7) - dimensionless time, defined in Eq. (7) - _mi Thiele modulus, defined in Eq. (7) - _i dimensionless concentration, defined in Eq. (7) - dimensionless radial position inside the bead, defined in Eq. (7) - _l0 initial dimension concentration of CPS-C, defined in Eq. (9), (10) - _i0 initial dimension concentration of component, defined in Eq. (9), (10) The authors wish to thank Dr. P. Gemeiner of Slovak Academy of Sciences for rendering of pectate gel. This work is supported by Ministry of Education (Grant No. 1/990 935/93).     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Excitability of Paramecium and the significance of negative surface charges

Summary On the basis of a model presented in a previous paper (Hook and Hildebrand, 1979) the influence of external cation concentrations [K⁺]₀, [Ca²⁺]₀ and of membrane voltage V_m (i.e. the actual potential difference between the two membrane faces) on the locomotor behavior of Paramecium is theoretically analyzed. In an extended model system we discuss the negative feedback of intraciliary calcium [Ca²⁺]_i on the excitability of the ciliary membrane. While a fast blocking of Ca channels is mediated by increased [Ca²⁺]_i and accounts for the short duration of action potentials, a slow [Ca²⁺ ]_i-dependent denaturation of channel molecules is assumed to determine excitability changes of Paramecium on a long time scale.It is emphasized that the duration of long-lasting ciliary reversal which reflects the excitability is not a direct function of the cation ratio J_u [K⁺]₀/[Ca²⁺] ₀ ^1/2 but rather of the membrane potential V_m.Introduction of negative surface charges can well explain why for a series of different [K⁺]₀, [Ca²⁺]₀ but constant J_a value the excitability is unchanged despite corresponding shifts in measured membrane potentials.     

Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca²⁺-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K_m for Ca ions of 0.4 M and V_max=12.5±2.4 mol P_i.mg^–1h^–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive³H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K_i=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high K_m) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca²⁺-ATPase - K-pNPPase onabain-sensitive, K⁺-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane     

Characterisation of [3H]-Darifenacin as a Novel Radioligand for the Study of Muscarinic M3 Receptors

Abstract

Darifenacin, (S)-2-[1-[2,3-dihydrobenzofuran-5-yl]-3-pyrrolidinyl]-2,2-diphenylacetamide, is a novel muscarinic M₃ antagonist. In this study we have compared the binding of [³H]-darifenacin to the five cloned human muscarinic receptors (m₁ - m₅) expressed in CHO cells. [³H]-darifenacin binds with 6 fold higher affinity to m₃ (K_D = 0.33 nmol/l) over m₁ (K_D = 1.6 nmol/l) receptors. There was no specific binding of [³H]-darifenacin to m₂ receptors and specific binding to m₄ and m₅ receptors was insufficient to determine a K_D. Binding of [³H]-darifenacin to m₁ and m₃ was displaced by atropine (m₁ pK_i = 9.36, m₃ pK_i = 9.4), 4-DAMP (m₁ pK_i = 9.04, m₃ pK_i = 9.19), pirenzepine (m₁ pK_i = 8.63, m₃ pK_i = 6.85), methoctramine (m₁ pK_i = 7.28, m₃ pK_i = 6.63), and darifenacin (m₁ pK_i = 8.36, m₃ pK_i = 9.14), demonstrating that [³H]-darifenacin represents the first selective m₃ radioligand.     

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO₂) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-¹³C]glucose, [U-¹³C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K _i=180 M) and uncompetitive (K_i=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K _i=1.3 M) with a low contribution of uncompetitive inhibition (K_i=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.     

Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate

Stirred tank (STR), bubble column (BCR) and airlift (ALR) bioreactors of 0.05 and 1.5 m³ total volume were compared for the production of 2,3-butanediol using Enterobacter aerogenes under microaerobic conditions. Batch fermentations were carried out at constant oxygen transfer rate (OTR=35 mmol/lh). At 0.05 m³ scale, the STR reactor achieved much higher biomass and product concentrations than the BCR and ALR reactors. At 1.5 m³ scale, however, exactly the same biomass and product concentrations could be obtained in both STR and ALR reactors. The 1.5 m³ ALR reactor performed also much better than its counterpart at small scale, achieving a productivity 2.4-fold as high as that of the 0.05 m³ BCL and ALR reactors. No differences in performances were observed between BCR and ALR. As compared to STR the tower reactors have a 12 time higher energetic efficiency (referred to product formation) and thus should be the choice for large scale production of 2,3-butanediol.The criterion of constant OTR or constant k _L a is not applicable for the scale-up of this oxygen-sensitive culture due to strong influence of reactor hydrodynamics under microaerobic conditions. The effects of mixing and circulation time on growth and metabolism of E. aerogenes were quantitatively studied in scaled-down experiments with continuous culture. For a successful scale-up of this microaerobic culture it is necessary to have an homogeneous oxygen supply over the entire reactor volume. Under conditions of inhomogeneous oxygen supply an optimum liquid circulation time exists which gives a maximum production of 2,3-butanediol.List of Symbols BD 2,3-butanediol - [mmol/l] saturation value of dissolved oxygen - D [h^–1] dilution rate - D [mm] reactor diameter - D _K [mm] top section diameter - D _R [mm] stirrer diameter - D _S [mm] draft tube diameter - EtOH ethanol - E _P [kg/kWh] energy efficiency refered to product formation - H [mm] height of reactor - HAc acetate - H _L [mm] height of liquid - k _L a [h^–1] volumetric oxygen transfer coefficient - N [rpm=min^–1] stirrer speed - OTR [mmol/lh] oxygen transfer rate - OUR [mmol/lh] oxygen uptake rate - p [Pa] pressure - P [kW] power input - P/V _L [kW/m³] specific power input - [mmHg] oxygen partial pressure (mmHg) or - [mmol/l] dissolved oxygen (mmol/l) - [mmol/gh] specific oxygen uptake rate - q _P [mmol/gh] specific productivity - R [Nm/kgK] gas constant, R = 287.06 - RQ respiration quotient - t _c [s] liquid circulation time - T [°C or K] temperature - TCA tricarboxylic acid - u _G [cm/s] mean superficial gas velocity - v _G [m/s] gas velocity at nozzels of gas distributor - V_G [l/h] aeration rate at inlet - V [m³ or l] total volume - V _L [m³ or l] liquid volume - V _N [l/mol] gas mole volume under normal conditions, V _N = 24.4116 - X [g/l] biomass concentration - CO₂ mole fraction in the effluent gas - O₂ mole fraction in the effluent gas - inlet (above the gas distributor) - ratio of oxygen consumed through TCA cycle to the total oxygen uptake rate - [g/l or kg/m³] density - [%] degree homogeneity - outlet of fermenter or top of the dispersion phase Dedicated to the 65th birthday of Professor Fritz Wagner.We thank Dr. C. Posten and T. Gabel for support with the computer control system UBICON. T.-G. Byun gratefully acknowledges financial support by DAAD.