Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Flight of the honey bee VI: energetics of wind tunnel exhaustion flights at defined fuel content,speed adaptation and aerodynamics

To gain information on extended flight energetics, quasi-natural flight conditions imitating steady horizontal flight were set by combining the tetheredflight wind-tunnel method with the exhaustion-flight method. The bees were suspended from a two-component aerodynamic balance at different, near optimum body angle of attack and were allowed to choose their own speed: their body mass and body weight was determined before and after a flight; their speed, lift, wingbeat frequency and total flight time were measured throughout a flight. These values were used to determine thrust, resultant aerodynamic force (magnitude and tilting angle), Reynolds number, total flight distance and total flight impulse. Flights in which lift was body weight were mostly obtained. Bees, flown to complete exhausion, were refed with 5, 10, 15 or 20 l of a 1.28-mol·l^-1 glucose solution (energy content w=18.5, 37.0, 55.5 or 74.0 J) and again flown to complete exhaustion at an ambient temperature of 25±1.5°C by a flight of known duration such that the calculation of absolute and relative metabolic power was possible. Mean body mass after exhaustion was 76.49±3.52 mg. During long term flights of 7.47–31.30 min similar changes in flight velocity, lift, thrust, aerodynamic force, wingbeat frequency and tilting angle took place, independent of the volume of feeding solution. After increasing rapidly within 15 s a more or less steady phase of 60–80% of total flight time, showing only a slight decrease, was followed by a steeper, more irregular decrease, finally reaching 0 within 20–30 s. In steady phases lift was nearly equal to resultant aerodynamic force; tilting angle was 79.8±4.0°, thrust to lift radio did not vary, thrust was 18.0±7.4% of lift, lift was somewhat higher/equal/lower than body mass in 61.3%, 16.1%, 22.6% of all totally analysable flights (n=31). The following parameters were varied as functions of volume of feeding solution (5–20 l in steps of 5 l) and energy content. (18.5–74.0 J in steps of 18.5 J): total flight time, velocity, total flight distance, mean lift, thrust, mean resultant aerodynamic force, tilting angle, total flight impulse, wingbeat frequency, metabolic power and metabolic power related to body mass, the latter related to empty, full and mean (=100 mg) body mass. The following positive correlations were found: L=1.069·10^-9 f ^2.538; R=1.629·10^-9 f ^2.464; P _m=7.079·10^-8 f ^2.456; P _m=0.008v+0.008; P _m=18.996L+0.022; P _m=19.782R+0.021; P _m=82.143T+0.028; P _m=1.245·bm _f ^1.424 ; P _{mrel e}=6.471·bm _f ^1.040 ; =83.248+0.385. The following negative correlations were found: V=3.939–0.032; T=1.324·10^-4–0.038·10^-4. Statistically significant correlations were not found in T(f), L(), R(), f(), P _m(bm _e), P _{m rel e}(bm _e), P _{m rel f}(bm _e), P _{m rel f}(bm _f).Abbreviations A(m²) frontal area - bl(m) body length - bm(mg) body mass - c(mol·1^-1) glucose concentration of feeding solution - c _D (dimensionless) drag coefficient, related to A - D(N) drag - F _w(N) body weight - F _wp weight of paper fragment lost at flight start - f wingbeat frequency (s^-1) - g(=9.81 m·s^-2) gravitational acceleration - I(Ns)=R(t) dt total impulse of a flight - L(N) lift vertical sustaining force component - P _m(J·s^-1=W) metabolic power - P_{m ret} (W·g^-1) metabolic power, related to body mass - R(N) resultant aerodynamic force - Re v·bl·v ^-1 (dimensionless) Reynolds number, related to body length - s(m) v(t) dt virtual flight distance of a flight - s(km) total virtual flight distance - T (N) thrust horizontal force component of horizontal flight - T _a (°C) ambient temperature - t(s) time - t _tot (s or min) total flight time - v(m·s^-1) flight velocity - v(l) volume of feeding solution - W (J) energy and energy content of V - ( °) body angle of attack between body longitudinal axis and flow direction - ( °) tilting angle ( 90°) between R and the horizont in horizontal flight v(=1.53·10^-5m²·s^-1 for air at 25°) kinematic viscosity - (=1.2 kg·m^-3 at 25°C) air density     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

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).     

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

The permeabilized cells of Trigonopsis variabilis CCY 15-1-3 having D-amino acid oxidase (DAAO) activity were used to convert cephalosporin C (CPS-C) into 7-(-ketoadipyl amido) cephalosporanic acid (CO-GL-7-ACA) in a batch bioreactor with good aeration and stirring during the process. The deacylation of 7--(4-carboxybutanamido)-cephalosporanic acid (GL-7-ACA) to 7-cephalosporanic acid (7-ACA) by permeabilized cells of Pseudomonas species 3635 having 4--(4-carboxybutamido)-cephalosporanic acid acylase (GL-7-ACA acylase) activity was performed in a batch bioreactor. A spectrophotometric method for the determination of CO-GL-7-ACA and 7-ACA was proposed. Experimental data were fitted by non-linear regression with parameters optimization. The sorption method (without reaction) was applied for the determination of cephalosporin effective diffusion coefficients in Ca-pectate gel beads. These beads were prepared by dropping a potassium pectate gel suspension of inactive permeabilized cells of Trigonopsis variabilis and Pseudomonas species, crosslinked with glutaraldehyde, into a stirred 0.2 M calcium chloride solution. Concentrations of appropriate cephem components were measured by the refractive method. Values of effective diffusion coefficients were calculated by the Fibbonacci optimization method.List of Symbols c _L mol/dm³ concentration on the surface of a bead - c _L0 mol/dm³ initial cephalosporin concentration - c _L mol/dm³ equilibrium cephalosporin concentration in the solution - c _s1 mol/dm³ concentration of CPS-C - c _s2 mol/dm³ concentration of GL-7-ACA - D _ei m²/s effective diffusion coefficient of the components - K _i mol/dm³ inhibition parameter in Eq. (2) - K _{m i} mol/dm³ Michaelis constant in Eq. (1) - K _{m 2} mol/dm³ Michaelis constant in Eq. (2) - n number of beads - q _n nonzero positive roots in Eq. (7) - r ₁ mol/(dm³·s) rate of the conversion of CPS-S to CO-GL-7-ACA - r ₂ mol/(dm³·s) rate of the conversion of GL-7-ACA to 7-ACA - R m radius of the bead - S( ) symbol for total residual sum of squares in Eq. (1) - t s time - V _{m 1} mol/(dm³·s) max. reaction rate in Eq. (1) - V _{m 2} mol/(dm³·s) max. reaction rate in Eq. (2) - V _L dm³ volume of the solution excluding the space occupied by beads - V _s dm³ volume of beads - y _i mol/(dm³ · s) symbol for experimental data in Eq. (1) - _i mol/(dm³· s) symbol for calculated data in Eq. (1) - _P porosity, defined by Eq. (5) - dimensionless parameter, defined by Eq. (6) 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)     

Intensity of microcarrier collisions in turbulent flow

A model is developed, allowing estimation of the share of inelastic interparticle collisions in total energy dissipation for stirred suspensions. The model is restricted to equal-sized, rigid, spherical particles of the same density as the surrounding Newtonian fluid. A number of simplifying assumptions had to be made in developing the model. According to the developed model, the share of collisions in energy dissipation is small.List of Symbols b parameter in velocity distribution function (Eq. (28)) - c _K factor in Kolmogoroff spectrum law (Eq. (20)) - D _t(r _p ) m²/s characteristic dispersivity at particle radius scale (Eq. (13)) - E(k, t) m³/s² energy spectrum as function of k and t (Eq. (16)) - E _K (k) m³/s² energy spectrum as function of k in Kolmogoroff-region (Eq. (20)) - E _p dimensionless mean kinetic energy of a colliding particle (Eq. (36)) - E _cp dimensionless kinetic energy exchange in a collision (Eq. (37)) - G(x, s) dimensionless energy spectrum as function of x and s (Eq. (16)) - G _B(x) dimensionless energy spectrum as function of x for boundary region (Eq. (29)) - G _K(x) dimensionless energy spectrum as function of x for Kolmogoroff-region (Eq. (21)) - g m/s² gravitational acceleration - I _cp dimensionless collision intensity per particle (Eq. (38)) - I _cv dimensionless volumetric collision intensity (Eq. (39)) - k l/m reciprocal of length scale of velocity fluctuations (Eq. (17)) - K dimensionless viscosity (Eq. (13)) - n(2) dimensionless particle collision rate (Eq. (12)) - n(r) l/s particle exchange rate as function of distance from observatory particle center (Eq. (7)) - r m vector describing position relative to observatory particle center (Eq. (2)) - r m scalar distance to observatory particle center (Eq. (3)) - r _pm particle radius (Eq. (1)) - s dimensionless time (Eq. (10)) - SC kg/ms³ Severity of collision (Eq. (1)) - t s time (Eq. (2)) - u(r, t) m/s velocity vector as function of position vector and time (Eq. (2)) - u(r, t) m/s magnitude of velocity vector as function of position vector and time (Eq. (3)) - u _r(r, t) m/s radial component of velocity vector as function of position vector and time (Eq. (3)) - u _r (r, t) m/s magnitude of radial component of velocity vector as function of position vector and time (Eq. (3)) - u (r, t) m/s latitudinal component of velocity vector as function of position vector and time (Eq. (3)) - u (r, t) m/s magnitude of latitudinal component of velocity vector as function of position vector and time (Eq. (3)) - u (r, t) m/s longitudinal component of velocity vector as function of position vector and time (Eq. (3)) - u (r, t) m/s magnitude of longitudinal component of velocity vector as function of position vector and time (Eq. (3)) - u _gsm/s superficial gas velocity - u(r) m/s root mean square velocity as function of distance from observatory particle center (Eq. (3)) - u_r(r) m/s root mean square radial velocity component as function of distance from observatory particle center (Eq. (4)) - u (r) m/s root mean square latitudinal velocity component as function of distance from observatory particle center (Eq. (4)) - u (r) m/s Root mean square longitudinal velocity component as function of distance from observatory particle center (Eq. (4)) - w(x) dimensionless root mean square velocity as function of dimensionless distance from observatory particle center (Eq. (11)) - V _pm³ particle volume (Eq. (36)) - w(2) dimensionless root mean square collision velocity (Eq. (34)) - w ^* parameter in boundary layer velocity equation (Eq. (24)) - x dimensionless distance to particle center (Eq. (9)) - x ^* value of x where G _Band G _K-curves touch (Eq. (32)) - x _K dimensionless micro-scale (Kolmogoroff-scale) of turbulence (Eq. (15)) - volumetric particle hold-up - m²/s³ energy dissipation per unit of mass - m²/s kinematic viscosity - kg/m³ density - (r) m³/s fluid-exchange rate as function of distance to observatory particle center - Latitudinal co-ordinate (Eq. (5)) - Longitudinal co-ordinate (Eq. (5))     

Synthetic substrates in the histochemical demonstration of intestinal disaccharidases

Summary The splitting of 6-Br-2-naphthyl-, -naphthyl-, and 4-Cl-5-Br-3-indolyl-glycosides which proved useful for the assessment of cytological localization of intestinal enzymes in previous studies was investigated using isolated human and rat intestinal disaccharidases as a source of enzyme activities.Previous findings based on histochemical studies were confirmed and extended. 6-Br-2naphthyl-D-glucoside is cleaved by glucoamylase and sucrase-isomaltase. The participatio of trehalase in splitting of this substrate is very low and can be neglected. The mentioned -glucosidases are responsible for the brush border staining of enterocytes with this substrate when unfixed cold microtome sections are used. Even when a differential heat inactivation of sucrase-isomaltase and of glucoamylase occurs during paraffin embedding (so that the staining in paraffin sections is due mostly to glucoamylase) the use of natural substrates is desirable for a more precise assessment of sucrase-isomaltase activity (but without the possibility of a correct localization).4-Cl-5-Br-3-indolyl--D-fucoside is the substrate of choice for the demonstration of lactase. Even when this substrate is split also by hetero--galactosidase and by acid (lysosomal) -galactosidase these activities do not disturb the histochemical demonstration of lactase. If however some doubts arise, the inhibition with p-Cl-mercuribenzoate (2 · 10^–4 M) is to be emloyed (lactase activity is not inhibited). Due to a low K_m and a high V_max of indolyl-fucoside and due to its extreme stability in solution (which enables to use the substrate solution repeatidly) this substrate is suitable in routine practice even though it is expensive. -naphthyl- and 4-Cl-5-Br-3-indolyl--D-glucosides are split by lactase and -glucosidase. Due to the fact that the mutual delineation of these activities is not easy and that K_m an V_max for lactase are not so favourable as in the case of fucoside these substrates are not recommended for the assessment of lactase.6-Br-2-naphthyl--D-glucoside is the substrate of choice for the histochemical studies concerned with hetero--galactosidase and 4-Cl-5-Br-3-indolyl--D-galactoside for acid -galactosidase.     

Phototactic responses in Haematococcus lacustris and its modification by light intensity and the carotenoid biosynthesis inhibitor Norflurazon

At fluence rates below 45 W· m^-2 cells of the flagellate stage of Haematococcus lacustris react only positively phototactically with a rather high degree of orientation (indicated by r values up to 0.66 with the Rayleigh test). The directedness of orientation decreases with decreasing irradiance. The degree of directedness of the phototactic response depends on the intensity of preirradiation: Low light intensity applied after strong light application results in a dark reaction (low r values), low light given after darkness stimulates a rather high degree of directedness of positive phototaxis. Weak blue light (=483 nm; 0.4 W · m^-2) stimulates positive phototactic response, whereas comparable red light (=658 nm; 0.5 W · m^-2) does not.Cells which were grown in a medium containing 10^-4 M Norflurazon (effective in inhibition of carotenoid biosynthesis) although maintaining motility completely lose the ability to react positively phototactically. The possible role of carotenoids in the phototactic orientation is discussed.     

A dynamic model of a two-synapse feedback loop in the vertebrate retina

The theoretical properties of synapses such as those in the retina which operate on graded potentials are developed using work on tetrodotoxin-treated synapses as a basis. A linearized model of a two-synapse negative feedback loop analogous to the bipolaramacrine feedback loop in the retina possesses a frequency response which developes an increasingly prominent resonance peak at higher input levels and under some circumstances shows instability. Psychophysical studies have shown that the visual system also exhibits this behaviour suggestive of progressive underdamping in a harmonic oscillator. Evidence in favor of the hypothesis that resonance originates in the loop is presented, the conclusions being that the loop functions to tune the retina to a range of temporal frequencies.Symbol Table V millivolts depolarisation relative to resting membrane potential - V _n , V _out pre-synaptic, post-synaptic depolarisation respectively - V _e , V _i reversal potential or e.m.f. of post-synaptic battery of excitatory, inhibitory synapses respectively - V _out (max) maximal post-synaptic depolarisation defined by Eq.(10c) - V ₀ input depolarisation for feedback loop - depolarisation potential normalised with respect to V _out(max) - I milliamperes of depolarising current - I _s post-synaptic membrane current - I _c cable current - I ₀ input depolarising current for feedback loop - I _max maximal physiological value for I ₀ =V _e ·G ₀ - i depolarising current normalised with respect to I_max - e reversal potential normalised with respect to V _e - r _i specific resistivity of internal medium - R _m membrane resistance - C _m membrane capacitance - cable space constant = R _m /2R _i - g ₀ characteristic cable conductance = 2/R _m ·R _i - G conductance of post-synaptic membrane - G _s maximal post-synaptic membrane conductance - g fraction of receptors occupied by transmitter = G/G _s - r the ratio G _s/G ₀ - membrane time constant = R _m·C_m - ₁ time constant of transmitter release in response to presynaptic depolarisation [Eq. (6)] - ₂ time constant of decay of g [Eq. (7a)] - _{2 2}·[1+k·exp(b·v _in)]^–1 - k equilibrium constant for transmitter-receptor interaction [Eq. (7a)] - b constant determining increase in rate of transmitter release with pre-synaptic depolarisation [Eq. (6)] - c concentration of transmitter in synaptic cleft normalised with respect to resting concentration - H _jk (s) linearised transfer function for synaptic transmission from neurone j to neurone k - G(s) H₁₂(s) - H(s) -H₂₁(s) - F(s) linearised closed-loop transfer function - _x 2 times spatial frequency of counterphase grating pattern - the ratio (1+s)/(_x)₂ - a the product (1+r)·k - d density of bipolars per unit area     

Model aided design of repeated fed-batch penicillin fermentation

Structured models of antibiotic fermentation that quantify maturation and aging of product forming biomass are fitted to experimental data. Conditions of superiority of repeated fed batch cultivation are characterized on the basis of a performance criterion that includes penicillin productivity and costs of operation. Emphasis is placed on the relevance of such research to the model aided design of optimal cyclic operation.List of Symbols c IU/mg cost factor - D s^–1 dilution rate - J IU · cm^–3 · h^–1 net productivity - k _p IU · mg^–11 · h^–1 specific product formation rate - k _pm IU · mg^–1 · h^–1 maximum specific product formation rate - p IU/cm³ concentration of penicillin - T s final time of fermentation - t s fermentation time - X kg/m³ concentration of biomass dry weight - X ₁kg/m³ concentration of young, immature biomass - X ₂ kg/m³ concentration of mature product forming biomass - X _c kg/m³ biomass concentration of the end of growth phase - X _mkg/m³ maximum biomass concentration Greek Letters s^–1 specific maturation rate - s^–1 specific aging rate - s^–1 specific growth rate - _m s^–1 maximum specific growth rate - _p s^–1 specific growth rate during the product formation phase - s cycle time - % volume fraction of draw-off Abbreviations CC chemostat culture - RFBC repeated fed batch culture - RBC repeated batch culture     

A simple dynamic method for on-line and off-line determination of kLa during cultivation of animal cells

Summary A new, fast method is described to determine k_La either off-line, or on-line during animal-cell cultivation. Since it does not need the equilibrium concentration of oxygen in the liquid phase (C*), it is not required to await a new steady state. Furthermore, the results do not depend on the calibration value of the dissolved-oxygen probe. The method yielded accurate values for k_La, both for an oxygen-consuming and a non-consuming system.Nomenclature C _L Dissolved-oxygen concentration [mol·m^-3] - C ^* C _L in equilibrium with the oxygen concentration in the gas phase [mol·m^-3] - C _L, Equilibrium oxygen concentration at stationary conditions [mol·m^-3] - k_La Volumetric oxygen transfer coefficient [s^-1] - r Specific oxygen consumption of biomass [mol·cell^-1·s^-1] - X Cell concentration [cells·m^-3] - t Time [s] - Noise of dissolved-oxygen probe [mol·m^-3] - Absolute error of k_La-measurement [s^-1]     

A theoretical and experimental evaluation of a novel radial-flow hollow fiber reactor for mammalian cell culture

A hollow fiber perfusion reactor constructed from pairs of concentric fibers forming a thin annular space is analyzed theoretically in terms of mass transfer resistances, and is shown experimentally to support the growth of an anchorage-dependent cell line in high-density culture. Hollow fiber perfusion reactors described in the literature typically employ a perfusion pathlength much greater than the distance that could be supported by diffusion alone, and analyses of these reactors typically incorporate the assumption of uniform perfusion throughout the cell mass despite many reported observations of inhomogeneous cell growth in perfusion reactors. The mathematical model developed for the annular reactor predicts that the metabolism of oxygen, carbon substrates, and proteins by anchorage-dependent cells can be supported by the reactor even in the absence of perfusion. The implications of nonuniform cell growth in perfusion reactors in general is discussed in terms of nutrient distribution. In the second part of the paper, the growth and metabolism of the mouse adrenal tumor line Y-1 in flask culture and in the annular reactor are compared. The reactor is shown to be a promising means for culturing anchorage-dependent cells at high density.List of Symbols c mol/dm³ substrate concentration - D _mm²/s effective diffusivity of substrate in the membrane - D _tm²/s effective diffusivity of substrate in the cell region - L _pm²s/kg hydraulic permeability of fiber - Pe _m Peclet number for membrane transport, _wR₁/D _m - Pe _t Peclet number for transport through cell mass, v _wR₂/D _t - Q mol/m³s zero-order consumption rate of substrate per unit volume of cell mass - r m radial distance from centerline of fiber lumen - R ₁, R ₂ m inner and outer radii of inner annular fiber (Fig. 1) - R ₃, ₄ m inner and outer radii of outer annular fiber (Fig. 1) - v _wm/s fluid velocity through the fiber wall at R ₁ - fraction of shell side filled with cells - dimensionless radial distance, R ₃/R₁ - dimensionless radial distance, R ₂/R ₁ - cm² hydraulic conductivity - viscosity - ², Thiele modulus - dimensionless radial distance, R ₄/R ₁     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Mixing-models applied to industrial batch bioreactors

Mixing models for bioreactors on the basis of the tanks-in-series concept are presented and a suitable parameter-estimation method is introduced. The Monte-Carlo-optimization procedure with the inhomogeneity-curve included in the objective function is used. Results of the parameter optimization procedure are given for stirred-tank-bioreactors equipped with one and three Rushton turbines under aerated conditions. The model designed for the stirredtank with three Rushton turbines is capable to describe the mixing properties, while in case of the stirred-tank with one Rushton turbine the simulated radial circulation time does not correlate with the measured one.List of Symbols a ₀₀...a _XY coefficients in Eq. (9) - d _i m stirrer diameter - D m tank diameter - E relative error - F _AX m³/s axial liquid flow rate - F _G m³/s aeration flow rate - F _RAD m³/s radial liquid flow rate - g m/s² acceleration of gravity - h _l m height of fluid in the tank - i _s(t) simulated inhomogeneity-curve - i _m(t) measured inhomogeneity-curve - k number of sensors - n 1/s stirrer revolutions - N number of tanks in the tanks-of-series-cascade - p number of measured time intervalls - t s time - t _c.AX s axial circulation time - t _c,RAD s radial circulation time - T _i °C temperature of sensors - T °C temperature at the end of the experiment - T ₀ °C temperature before pulse injection - V _tot m³ total liquid volume - V _C m³ liquid volume of circulation cascade, additional index specifications describe the cascade elements (Figs.1 and 2) - V _M m³ liquid volume of well mixed stirrer compartment - w ₀ m/s superficial gas velocity - X, Y exponents in eq. (9) - kg/m³ density - Pas dynamic viscosity - m²/s kinematic viscosity - s time constant (time for 63,2% of T ) of the signal Dimensionless Numbers stirrer Froude number - aeration Froude number     

Oxygen transfer in a stirred loop fermentor with dilute polymer solutions

Oxygen transfer in a 0.35 m diameter stirred loop fermentor (a stirred tank with a concentric draft tube) has been studied with water containing a small amount of polymer(polyethylene oxide) as a drag-reducing additive.Power consumption was measured. It was found that the addition of polyethylene oxide causes an increase of power consumption. This is contrary to the results reported in the literature.Volumetric mass transfer coefficients (K _La) were measured. In water the introduction of the draft tube increased the K _La coefficient. The increase in K _La became larger with impeller speed. On the other hand, mass transfer in dilute polymer solutions decreased due to the presence of the draft tube. An empirical correlation has been proposed for the volumetric mass transfer coefficient in stirred loop fermentors. It has a general applicability.List of Symbols a 1/m specific surface area - C constant in Eq. (6) - g m/s² gravitational acceleration - K _L m/s overall liquid-phase mass transfer coefficient - n 1/s impeller speed - P W aerated power input by mechanical agitation - P _g W power input by sparged air - Q m³/min volumetric gas flow rate - U _sg m/s superficial gas velocity - V m³ liquid volume Greek Symbols exponents in Eq. (3) - exponent in Eq. (6) - kg/m³ density     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity     

Chromosomal polymorphisms involving telomere fusion,centromeric inactivation and centromere shift in the ant Myrmecia (pilosula) n=1

Detailed karyological surveys of the ant Myrmecia pilosula species group, which is characterized by the lowest chromosome number in higher organisms (2n=2), were attempted. We revealed that this species has developed highly complicated chromosomal polymorphisms. Their chromosome numbers are in the range 2n=2, 3, and 4, and six polymorphic chromosomes are involved, i.e., two for chromosome 1 (denoted as SM₁ and ST₁), three for chromosome 2 (A₂, A₂, and M₂), and M₍₁₊₂₎ for the 2n=2 karyotype. We suggested that these chromosomes were induced from a pseudo-acrocentric (A ₁ ^M ) and A₂ as follows: (1) A ₁ ^M SM₁ or ST₁ by two independent pericentric inversions; (2) A₂A₂M₂ by chromosomal gap insertion and centromere shift; and (3) ST₁+A₂M₍₁₊₂₎ by telomere fusion, where (3) means that the 2n=2 karyotype was derived secondarily from a 2n=4 karyotype. It is a noteworthy finding that active nucleolus organizer (NOR) sites, in terms of silver staining, are tightly linked with the centromere in this species, and that both the centromere and NOR of A₂ were inactivated after the telomere fusion.     

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.Our present study concentrates on development of procedures for describing the performance of devices where enzyme-catalyzed reactions between two substrates take place, and for the rational design and optimization of the reactors considered. Within this context, an analytical model was developed for immobilized enzyme packed-bed reactors; it takes into account internal diffusion limitations for the cosubstrates, and hydrodynamic backmixing effects. In order to overcome the complex mathematical problems involved, the compartmental analysis approach was employed.Using this model, performance was simulated for various configurations of the enzymic unit, i.e. from a continuously operated stirred tank reactor (CSTR) to an essentially plug flow type. In addition, an experimental method is described for quantitatively assessing the backmixing effects prevailing in the reactor.The procedures established also provide the ground for further developments, particularly for systems where, in parallel to the enzymic reaction, additional processes (e. g. complexation) take place.List of Symbols C _j,i mM Concentration of substrate j in the pores of stage - iD _j cm²/s Internal (pore) diffusion coefficient of substrate j; defined in Eq. (7) - D _e cm²/s Axial dispersion diffusion coefficient - D _j, cm²/s cm²/s Bulk diffusion coefficient for substrate j - E mM Enzyme concentration inside the catalytic pores - J _j,immol/s/cm² Net flux of substrate j taking place from the bulk of stage i into the corresponding pores; defined in Eq. (6) - K _m,1, K _m,2 mM Michaelis-Menten constants for cosubstrates 1 and 2, respectively - k s ^–1 Catalytic constant - k _s cm/s Catalytic constant - n Total number of elementary stages in the reactor - Q cm³/s Volumetric flow rate throught the reactor - r cm Radius of the pore - R _j,i mM/s Reaction rate of substrate j in stage i, in terms of volumetric units - S cm² Internal surface of a pore - S _j,0 mM Concentration of substrate j in the reactor feed - S _j,i–1, S _j,i mM Concentration of substrate j in the bulk phase leaving stages i — 1 and i, respectivley - V _i cm³ Total volume of stage i (bulk phase + pore phase + inert solid carrier) - V cm³ Total volume of the reactor - V _m ^* mmol/s/cm² Maximal reaction rate in terms of surface units; defined in Eq. (8) - V _m mM/s Maximal reaction rate in terms of volumetric units; defined in Eq. (8) - V _p cm³ Volume of one pore - y cm Axial coordinate of the pores - y ₀ cm Depth of the pores - Z cm Axial coordinate of the reactor - Z ₀ cm Length of the reactor - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - Ratio between the radius of the enzyme molecule and the radius of the pore (dimensionless) - V₁ Dimensionless parameter; defined in Eq. (21) - v₂ Dimensionless parameter; defined in Eq. (21) - Q Volumetric packing density of catalytic particles (dimensionless) - Ø Porosity of the catalytic particles (dimensionless) - Ø Dimensionless concentration of substrate j in pores of stage i; defined in Eq. (16) - _j,i-1,_j,i Dimensionless concentration of substrate j in the bulk phase of stage i; defined in Eq. (18) - Dimensionless position; defined in Eq. (16) - ² s² Variance; defined in Eq. (33) - Mean residence time in the reactor; defined in Eq. (33)     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α 2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by -adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·l^-1 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3,5-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol·l^-1 and naphazoline at 1 mol·l^-1 inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate in the cells in both the presence and the absence of isoproterenol, a -adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3,5-monophosphate, but only when they were present at relatively high concentrations (above 100 mol·l^-1). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3,5-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 g·ml^-1) for 15 h or treatment with 100 nmol·l^-1 yohimbine (an ₂-adrenergic antagonist) inhibited the effects of the -adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate, but prazosin (an ₁adrenergic antagonist) at 100 nmol·l^-1 was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase, which occurs as result of stimulation of a pathway that involves ₁adrenergic and a inhibitory GTP-binding protein.Abbreviations A-kinase cAMP-dependent protein kinase - BSS balanced salts solution - CaM calmodulin - cAMP cyclic adenosine-3,5-monophosphate - Clo clonidine - EDTA ethylenediaminetetra-acetic acid - G-protein GTP-binding protein - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate Mex, methoxamine - MSH melanocyte-stimulating hormone - Nap naphazoline - NE norepinephrine - Oxy oxymetazoline - Phe phenylephrine - PTX pertussis toxin