The effects of fermentation conditions on yeast cell debris particle size distribution during high pressure homogenisation

Experiments were performed to characterize the particle size distribution of bakers' yeast cells during high pressure homogenisation. Results were obtained for mechanically agitated batch and continuously grown cultures under a range of operating conditions. It was found that the dependency of cell debris size distribution on the number of passes through the homogeniser and the homogeniser pressure was independent of the cell properties and culture conditions, but for a fixed pressure and number of passes the extent of disruption was strongly affected by the operating conditions in the fermenter. The entire cell debris size distributions were successfully simulated using the mean and variance of the distributions and a previously published model equation which related these parameters to the operating pressure and number of passes through the homogeniser.List of Symbols k breakage coefficient in Eq. 1 - d cell diameter - d ₅₀ median diameter of homogenate size distribution - d ₅₀ dimensionless d ₅₀ defined as - D dilution rate - F(d _NP) cumulative undersize distribution (volume basis) - N number of passes - P total pressure - P _threshold threshold pressure - P (P-P _threshold) - w Boltzmann parameter, Eq. 4 - w dimensionless standard deviation defined as Greek Letters exponent in Eq. 1 - exponent in Eq. 1 UCL is the Biotechnology and Biological Sciences Research Council's Interdisciplinary Research Centre for Biochemical Engineering and the Council's support to the participating UCL departments is gratefully acknowledged. The provision of continuous fermentation material from Dr. M. Gregory, Process System Engineering IRC, is gratefully acknowledged.     

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).     

Comparison of three granular support materials for anaerobic fluidized bed systems

Summary Three different materials, kaolin, pozzolana and biolite (a material used in a commercial anaerobic fluidized bed treatment process) when tested as supports for an anaerobic fluidized bed system had similar physical and fluidization properties but behaved differently towards the biomass hold-up. However, all three systems attained similar removal efficiency rates.Nomenclature U Fluidization velocity (m/s) - U₁ Terminal fluidization velocity (m/s) - g Local acceleration due to gravity (m/s²) - _s Solid density (kg/m³) - _f Fluid density (kg/m³) - P Pressure drop (Pa) - HRT Hydraulic retention time (days) - H_mf Height of bed at minimum fluidization (m) - H Height of bed (m) - C_d Drag coefficient (dimensionless) - W Mass of solids in bed (kg) - dp Particle diameter (m) - A Cross-sectional area of column (m²) - h column height (m) - Rc_t Terminal Reynolds no. - Voidagc (fractional free volume, dimensionless) - _mf Voidage (fractional free volume) at minimum of fluidization (dimensionless)     

Data consistency,yield and maintenance coefficient for the growth of Candida lypoytica and Pseudomonas aeruginosa on n-hexadecane

Summary When more than the minimum number of variables are measured, and measurement error is taken into account, the results of parameter estimation depend on which of the measured variables are selected for this purpose. The reparameterization of Pirt's models for growth produces multiresponse models with common parameters. By using the covariate adjustment technique, a unit variate linear model with covariates is obtained. This allows a combined point and interval estimates of biomass energetic yield and maintenance coefficient to be obtained using standard multiple regression programmes. When this method was applied using form I and form II of the Pirt's models, good combined estimates were obtained and compared. Using data from the literature for Candida lipolytica produced reliable results. However, for Pseudomonas aeruginosa, which has been known to produce intermediate products, a modified Pirt's model is required for a good estimate of the biomass energetic yield.Nomenclature a Mole of ammonia per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - b Moles of oxygen per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - c Moles of water per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon; no of covariates included in model - d Moles of carbon dioxide per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - e _i Error terms in Eqs. (6–8) - l Atomic ratio of oxygen to carbon in organic substrate, dimensionless - m Atomic ratio of hydrogen to carbon in organic substrate, dimensionless - m _e Rate of organic substrate consumption for maintenance, g equiv. of available electrons in biomass (h) or kcal/Kcal of biomass(h) - n Atomic ratio of oxygen to carbon in biomass, dimensionless - p Atomic ratio of hydrogen to carbon in biomass, dimensionless - Q _{CO 2} Rate of evolution of carbon dioxide, g moles/g dry wt (h) - Q _{O 2} Rate of oxygen consumption, g moles/g dry wt (h) - Q _s Rate of organic substrate consumption g/g dry wt (h) - q Atomic ratio of nitrogen to carbon in biomass, dimensionless - r Atom ratio of hydrogen to carbon in products, dimensionless; the number of parameters of interest - s Atomic ratio of oxygen to carbon in products, dimensionless - t Atomic ratio of nitrogen to carbon in products, dimensionless - r Mean of k responses in Eq. (10) - x _ki Kth response in the ith observation - y _c Biomass carbon yield (fraction of organic substrate carbon in biomass), dimensionless - z _i Covariate matrix - z Fraction of organic substrate carbon in products, dimensionless - a _i Parameters associated with covariates - _s Reductance degree of biomass, equivalents of available electrons per gram atom carbon - Reductance degree of organic substrate, equivalents of available electrons per gram atom carbon - Fraction of energy in organic substrate which is evolved as heat, dimensionless - Fraction of available electrons transferred to biomass; biomass energetic yield - True growth yield - Specific growth rate, h^-1 - _p Fraction of available electrons incorporated into products; product energetic yield - Correlation coefficient - Mass fraction carbon - ² Mean square error of model (10)     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Enhancement of protein precipitate strength and density by low-frequency conditioning

The use of a continuous, low-frequency conditioning process to alter the structure of protein precipitate aggregates is examined. An increase in the density of aggregates is correlated with the levels of fluid acceleration and hence hydrodynamic stress to which the aggregates are exposed during conditioning. A combination of low-frequency conditioning followed by shear break-up (as in the feed zone to a high-speed disk-stack centrifuge) is shown to result in a precipitate suspension of increased particle size at the fine end of the distribution, and having a greater sedimentation velocity. The resistance of large aggregates to shear disruption is increased by low-frequency conditioning.List of Symbols CR conditioning ratio - CRS conditioning ratio after shearing - d m amplitude of displacement - D m particle size - D _c m critical size for centrifuge recovery - f s^–1 frequency of vibration - G s^–1 mean velocity gradient - Q m³/s volumetric throughput - SR shear ratio - t s ageing time Greek Symbols s^–1 mass-average shear rate - K sedimentation shape factor - _a kg/m³ aggregate density - _f kg/m³ fluid density - _s kg/m³ solids density - kg/m³ aggregate-suspension density difference - Ns/m² kinematic viscosity - amplitude of pulse ratio (ref. 23, 9) - s mean residence time - _s solids volume fraction     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Cloned T cells recognize Trichinella spiralis antigen in association with an Ek βEk α restriction element

A method is described for the production of T-cell lines and clones specific for solubilized Trichinella spiralis antigens. hese T cells are antigen-specific and do not respond to challenge with a third party antigen (lysozyme). The proliferation responses of the cloned T cells are specifically inhibited by anti-I-E but not by anti I-A subregion monoclonal reagents. The inhibition patterns obtained are consistent with cis-gene complementation in B10.K cells involving the E^k -chain and the E^k -chain of the I-E molecule. Inhibition is obtained with an E^k -specific monoclonal antibody (H9-14.8) but not with an A^k -specific monoclonal antibody (10-2.16). Inhibition was also observed with Ia.7-specific (H40-242) or Ia.22-specific (17-3-3) monoclonal antibodies. The inhibition patterns were confirmed by antigen presentation experiments using recombinant inbred mice. Only B 10.K (E^k E^k spleen cells and not B 10.A(5R) (E^b E^k ) or B10.S(9R) (E^s E^k ) spleen cells could effectively present T. spiralis antigens. The role of hybrid Ia molecules in the immune response to T. spiralis is discussed.     

On the appropriateness of use of a continuous formulation for the modelling of discrete multireactant systems following Michaëlis–Menten kinetics

The possibility of solving the mass balances to a multiplicity of substrates within a CSTR in the presence of a chemical reaction following Michaelis-Menten kinetics using the assumption that the discrete distribution of said substrates is well approximated by an equivalent continuous distribution on the molecular weight is explored. The applicability of such reasoning is tested with a convenient numerical example. In addition to providing the limiting behavior of the discrete formulation as the number of homologous substrates increases, the continuous formulation yields in general simpler functional forms for the final distribution of substrates than the discrete counterpart due to the recursive nature of the solution in the latter case.List of Symbols C{N. M} mol/m³ concentration of substrate containing N monomer residues each with molecular weight M - {N, M} normalized value of C{N. M} - C {M} mol/m³ da concentration of substrate of molecular weight M - _in normalized value of C {M} at the i-th iteration of a finite difference method - {M} normalized value of C {M} - C ₀{N.M} mol/m³ inlet concentration of substrate containing N monomer residues each with molecular weight M - {N ·M} normalized value of C₀{N. M} - _{0 i} normalized value of C ₀ {M} at the i-th iteration of a finite difference method - C ₀ {M} mol/m³ da initial concentration of substrate of molecular weight M - C _tot mol/m³ (constant) overall concentration of substrates (discrete model) - C _tot mol/m³ (constant) overall concentration of substrates (continuous model) - D deviation of the continuous approach relative to the discrete approach - i dummy integer variable - I arbitrary integration constant - j dummy integer variable - k dummy integer variable - K _m mol/m³ Michaëlis-Menten constant for the substrates - l dummy integer variable - M da molecular weight of substrate - M normalized value of M - M da maximum molecular weight of a reacting substrate - N number of monomer residues of a reacting substrate - N maximum number of monomer residues of a reacting substrate - N total number of increments for the finite difference method - Q m³/s volumetric flow rate of liquid through the reactor - S inert product molecule - S _i substrate containing i monomer residues - V m³ volume of the reactor - v _max mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate - v _max{N. M} mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate containing N monomer residues with molecular weight M - _max{N · M} dimensionless value of v_max{N. M} (discrete model) - _max{M} dimensionless value of v _max {M} (continuous model) - mol/m³ s molecular weight-averaged value of v_max (discrete model) - mol.da/m³s molecular weight-averaged value of v_max (continuous model) - v _max {M} mol.da/m³s reaction rate under saturating conditions of the enzyme active site with substrate with molecular weight M - _max {M} dimensionless value of v_max{M} - _{max, (i)} dimensionless value of v_max{M} at the i-th iteration of a finite difference method - v _max mol/m³ s reference constant value of v _max Greek Symbols dimensionless operating parameter (discrete distribution) - dimensionless operating parameter (continuous distribution) - M da (average) molecular weight of a monomeric subunit - M selected increment for the finite difference method - auxiliary corrective factor (discrete model)     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).