C-phycocyanin and allophycocyanin in two species of blue-green algae

1. The biliproteins C-phycocyanin and allophycocyanin were purified from the blue-green alga Anabaena variabilis by ammonium sulphate fractionation and gel filtration. 2. An assay procedure that enabled the proportion of the two pigments, present as a mixture, to be determined was devised by using the data provided by spectrophotometric analysis of the purified biliproteins. 3. The degree of association and relative proportions of the two pigments were analysed by the application of this procedure to the components separated by thin-layer gel filtration. 4. The C-phycocyanin/allophycocyanin ratio remained essentially constant in algal extracts prepared at various stages throughout the growth cycle or after growth under conditions of reduced illumination. 5. The behaviour of the C-phycocyanin aggregate species from Anacystis nidulans suggested that they were of appreciably lower molecular weight than those observed in extracts of Anabaena variabilis.     

Constant specific growth rate in fed-batch cultivation of Bordetella pertussis using adaptive control

Monitoring and control of production processes for biopharmaceuticals have become standard requirements to support consistency and quality. In this paper, a constant specific growth rate in fed-batch cultivation of Bordetella pertussis is achieved by a newly designed specific growth rate controller. The performance of standard control methods is limited because of the time-varying characteristics due to the exponentially increasing biomass and volume. To cope with the changing dynamics, a stable model reference adaptive controller is designed which adapts the controller settings as volume and biomass increase. An important asset of the design is that dissolved oxygen is the only required online measurement. An original design without considering the dissolved oxygen dynamics resulted experimentally in oscillatory behaviour. Hence, in contrast to common believes, it is essential to include dissolved oxygen dynamics. The robustness of this novel design was tested in simulation. The validity of the design was confirmed by laboratory experiments for small-scale production of B. pertussis. The controller was able to regulate the specific growth rate at the desired set point, even during a long fed-batch cultivation time with exponentially increasing demands for substrates and oxygen.     

Physicochemical properties of the microbial exopolysaccharide ethapolan synthesized on a mixture of growth substrates

Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on single substrates. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3:2:1:1. EPS with a higher proportion of fatty acids was synthesized during growth on the mixture of ethanol and glucose. Average molecular weight and the proportion of high-molecular (over two million) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H+ form, and in the Cu(2+)-glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.     

Mechanism of protection of peroxidase activity by oscillatory dynamics.

The peroxidase-oxidase reaction is known to involve reactive oxygen species as intermediates. These intermediates inactivate many types of biomolecules, including peroxidase itself. Previously, we have shown that oscillatory dynamics in the peroxidase-oxidase reaction seem to protect the enzyme from inactivation. It was suggested that this is due to a lower average concentration of reactive oxygen species in the oscillatory state compared to the steady state. Here, we studied the peroxidase-oxidase reaction with either 4-hydroxybenzoic acid or melatonin as cofactors. We show that the protective effect of oscillatory dynamics is present in both cases. We also found that the enzyme degradation depends on the concentration of the cofactor and on the pH of the reaction mixture. We simulated the oscillatory behaviour, including the oscillation/steady state bistability observed experimentally, using a detailed reaction scheme. The computational results confirm the hypothesis that protection is due to lower average concentrations of superoxide radical during oscillations. They also show that the shape of the oscillations changes with increasing cofactor concentration resulting in a further decrease in the average concentration of radicals. We therefore hypothesize that the protective effect of oscillatory dynamics is a general effect in this system.     

Model for calcium dependent oscillatory growth in pollen tubes

Experiments have shown that pollen tubes grow in an oscillatory mode, the mechanism of which is poorly understood. We propose a theoretical growth model of pollen tubes exhibiting such oscillatory behaviour. The pollen tube and the surrounding medium are represented by two immiscible fluids separated by an interface. The physical variables are pressure, surface tension, density and viscosity, which depend on relevant biological quantities, namely calcium concentration and thickness of the cell wall. The essential features generally believed to control oscillating growth are included in the model, namely a turgor pressure, a viscous cell wall which yields under pressure, stretch-activated calcium channels which transport calcium ions into the cytoplasm and an exocytosis rate dependent on the cytosolic calcium concentration in the apex of the cell. We find that a calcium dependent vesicle recycling mechanism is necessary to obtain an oscillating growth rate in our model. We study the variation in the frequency of the growth rate by changing the extracellular calcium concentration and the density of ion channels in the membrane. We compare the predictions of our model with experimental data on the frequency of oscillation versus growth speed, calcium concentration and density of calcium channels.     

Use of cell cycle analysis to characterise growth and interferon-γ production in perfusion culture of CHO cells

The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intensification of synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S grown on a mixture of substrates

Enhanced synthesis of the exopolysaccharide (EPS) ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1:1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3-0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C2-metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture was 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4 to 7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

The dynamical analogy between microbial growth on mixtures of substrates and population growth of competing species

There is a similarity between the metabolic dynamics of a microbial species growing on a mixture of two substrates and the dynamics of growth of two competing populations. Specifically, the enzymes catalyzing the uptake and catabolism of substrates exhibit phenomena analogous to extinction and coexistence."Extinction" of the enzymes associated with one of the substrates results in sequential utilization of the substrates (diauxie) (Monod, 1942). "Coexistence" of the enzymes associated with the substrates results in simultaneous utilization of the substrates (Egli, 1995). Here, we formulate a simple model that shows the basis for this dynamical similarity: The equations describing the evolution of the enzyme levels are dynamical analogs of the Lotka-Volterra model for two competing species. The analogy suggests ways of capturing the experimentally observed preculture-dependent growth patterns, i.e., growth patterns that vary depending on the physiological state of the preculture.     

Improving effects obtained by the ovariectomy or treatment with tamoxifen of female diabetic rats over the function and enzyme activities of liver mitochondria.

In the present work we studied, in female chronic diabetic rats the effect of either the parenteral administration of tamoxifen (TAM) (500 micrograms.kg-1.day-1) for 15 days or the ovariectomy upon the respiration and oscillatory behaviour of intact mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cox) of disrupted liver mitochondria. The treatment with TAM as well as the ovariectomy of diabetic animals significantly increased the respiratory control (RC) and the state 3 (S3) of respiration of intact liver mitochondria with the three substrates assayed (3-hydroxybutyrate, malate-glutamate and succinate). Both treatments also lowered significantly the damped factors of the oscillatory variation of liver intact mitochondria of diabetic rats. Moreover, the two above-mentioned treatments restored the activities of HBD and Cox of liver disrupted mitochondria to normal values. The effect of estrogens at level of its receptors in the modulation of liver mitochondrial function and liver HBD and Cox activities in chronic diabetes is discussed.     

Mixture enhances productivity in a two-species forest: evidence from a modeling approach

The effect of mixture on productivity has been widely studied for applications related to agriculture but results in forestry are scarce due to the difficulty of conducting experiments. Using a modeling approach, we analyzed the effect of mixture on the productivity of forest stands composed of sessile oak and Scots pine. To determine whether mixture had a positive effect on productivity and if there was an optimum mixing proportion, we used an aggregation technique involving a mean-field approximation to analyze a distance-dependent individual-based model. We conducted a local sensitivity analysis to identify the factors that influenced the results the most. Our model made it possible to predict the species proportion where productivity peaks. This indicates that transgressive over-yielding can occur in these stands and suggests that the two species are complementary. For the studied growth period, mixture does have a positive effect on the productivity of oak-pine stands. Depending on the plot, the optimum species proportion ranges from 38 to 74% of oak and the gain in productivity compared to the current mixture is 2.2% on average. The optimum mixing proportion mainly depends on parameters concerning intra-specific oak competition and yet, intra-specific competition higher than inter-specific competition was not sufficient to ensure over-yielding in these stands. Our work also shows how results obtained for individual tree growth may provide information on the productivity of the whole stand. This approach could help us to better understand the link between productivity, stand characteristics, and species growth parameters in mixed forests.     

Boolean analysis of cell regulation networks

A comparison is made between the predictions of the Boolean and continuous analysis of a regulation model when the formation of two mediators interacting by cross-inhibition is stimulated by one or two specific signals. For such a system, the Boolean analysis reproduces the characteristics of behaviour previously predicted by continuous analysis (multiple stable states of opposite type, discontinuous transition, and associated hysteresis phenomenon). The qualitative agreement between the two methods allows a qualitative but rigorous treatment of regulation systems in which the Boolean analysis is applicable. From a general schematic representation of interaction in bidirectional control systems, we analyse by the Boolean method a large range of possible systems of increasing complexities which could theoretically apply. Previously unforeseen consequences of some systems are described. After that, we give a logical analysis of a well-known system (negative loop grafted with additional external controls) and discuss the application of such a system to explain certain oscillatory phenomena in the cell, showing the disrupting role of an additional control on the expected behaviour. Thus, when the analysis of a model including a negative loop does not indicate the possibility of experimentally suggested oscillations, we propose other simple logical structures which can predict this behaviour. Finally, we show a logical analysis of an opposite type of example of cell regulation where the biochemical observations can be accounted for simply by a negative loop grafted with one input variable.     

Enhanced Synthesis of the Exopolysaccharide Ethapolan by Acinetobacter sp. 12S Grown on a Mixture of Substrates

Enhanced synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1 : 1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3–0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C₂ metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture were 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4–7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

Nature and significance of oscillatory behavior during solvent production by Clostridium acetobutylicum in continuous culture

The concurrent production of acids and solvents and the production of acetone during continuous culture in a product-limited chemostat indicated that the culture contained a mixture of acid- and solvent-producing cells. Periodic oscillations in the yield of end products and the specific growth rate of the culture were ob served during undisturbed continuous culture at a constant dilution rate. The increased specific growth rate was associated with an increased acid yield and an increase in the rate of cell division and the proportion of short rods. The decreased specific growth rate was as sociated with an increase in the solvent yield and a decrease in the rate of cell division, resulting in the production of elongated rods. It is proposed that the oscillatory behavior observed during continuous culture is an inherent characteristic related to the shift from primary to secondary metabolism. A major consequence of the oscillation of the specific rates of growth and division in cultures containing acid- and solvent-producing cells is that it precludes the attainment of a true steady state during continuous culture.     

Kinetic model for dynein oscillatory activity

A kinetic model for dynein, a molecular motor, is considered. This model explains the oscillatory behaviour, observed by Chikako Shingyoji et al. [Ch. Shingyoji, H. Higuchi, M. Yoshimura, E. Katayama, T. Yanagida, Dynein arms are oscillatory force generators, Nature 393 (1998) 711-714.] and by Susumu Aoyama and Ritsu Kamiya [S. Aoyama, R. Kamiya, Cyclical interactions between two outer doublet microtubules in split flagellar axonemes, Biophys. J. 89 (2005) 3261-3268.] in surprisingly simple axonemal fragments. The model shows that sustained oscillations can be generated due to the obligate cooperative interaction of the two dynein heads in the axonemal fragments. No other feedback control interactions are involved in the model to explain oscillations, similar to those observed experimentally, for realistic dynein rate constants. The modified model shows how the ATP hydrolytic exhaustion influences the amplitude and frequency of dynein oscillatory activity.     

Investigation of bacterial growth on mixed substrates: experimental evaluation of cybernetic models

Cybernetic models, developed earlier by the authors, have been evaluated experimentally for the growth of Klebsiella oxytoca in batch cultures using mixed substrates from glucose, xylose, arabinose, lactose, and fructose. Based entirely on information procured from batch growth on single substrates, the models accurately predict without further parameter fitting, diauxic growth on mixed substrates, automatically predicting the order in which the substrates are consumed. Even triauxic growth on a mixture of glucose, xylose, and lactose is predicted by the model based on single substrate data. Growth on glucose-fructose mixtures appears to need a slightly modified strategy for cybernetic variables.     

Hypothesis Testing in a Mixture Case–Control Model

Summary We consider a problem of testing mixture proportions using two‐sample data, one from group one and the other from a mixture of groups one and two with unknown proportion, λ, for being in group two. Various statistical applications, including microarray study, infectious epidemiological studies, case–control studies with contaminated controls, clinical trials allowing “nonresponders,” genetic studies for gene mutation, and fishery applications can be formulated in this setup. Under the assumption that the log ratio of probability (density) functions from the two groups is linear in the observations, we propose a generalized score test statistic to test the mixture proportion. Under some regularity conditions, it is shown that this statistic converges to a weighted chi‐squared random variable under the null hypothesis of λ= 0 , where the weight depends only on the sampling fraction of both groups. The permutation method is used to provide more reliable finite sample approximation. Simulation results and two real data applications are presented.     

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.     

Role of substrate inhibition kinetics in enzymatic chemical oscillations.

Two chemical kinetic models are investigated using standard nonlinear dynamics techniques to determine the conditions under which substrate inhibition kinetics can lead to oscillations. The first model is a classical substrate inhibition scheme based on Michaelis-Menten kinetics and involves a single substrate. Only when this reaction takes place in a flow reactor (i.e., both substrate and product are taken to follow reversible flow terms) are oscillations observed; however, the range of parameter values over which such oscillations occur is so narrow it is experimentally unobservable. A second model based on a general mechanism applied to the kinetics of many pH-dependent enzymes is also studied. This second model includes both substrate inhibition kinetics as well as autocatalysis through the activation of the enzyme by hydrogen ion. We find that it is the autocatalysis that is always responsible for oscillatory behavior in this scheme. The substrate inhibition terms affect the steady-state behavior but do not lead to oscillations unless product inhibition or multiple substrates are present; this is a general conclusion we can draw from our studies of both the classical substrate inhibition scheme and the pH-dependent enzyme mechanism. Finally, an analysis of the nullclines for these two models allows us to prove that the nullcline slopes must have a negative value for oscillatory behavior to exist; this proof can explain our results. From our analysis, we conclude with a brief discussion of other enzymes that might be expected to produce oscillatory behavior based on a pH-dependent substrate inhibition mechanism.     

Intermittent control models of human standing: similarities and differences

Two architectures of intermittent control are compared and contrasted in the context of the single inverted pendulum model often used for describing standing in humans. The architectures are similar insofar as they use periods of open-loop control punctuated by switching events when crossing a switching surface to keep the system state trajectories close to trajectories leading to equilibrium. The architectures differ in two significant ways. Firstly, in one case, the open-loop control trajectory is generated by a system-matched hold, and in the other case, the open-loop control signal is zero. Secondly, prediction is used in one case but not the other. The former difference is examined in this paper. The zero control alternative leads to periodic oscillations associated with limit cycles; whereas the system-matched control alternative gives trajectories (including homoclinic orbits) which contain the equilibrium point and do not have oscillatory behaviour. Despite this difference in behaviour, it is further shown that behaviour can appear similar when either the system is perturbed by additive noise or the system-matched trajectory generation is perturbed. The purpose of the research is to come to a common approach for understanding the theoretical properties of the two alternatives with the twin aims of choosing which provides the best explanation of current experimental data (which may not, by itself, distinguish beween the two alternatives) and suggesting future experiments to distinguish beween the two alternatives.