Dynamic characteristics of transient responses

Transient responses of signaling molecules are seen in a wide variety of cellular processes that are mediated by distinct molecular mechanisms. Although transient responses might intuitively be thought to depend on the absolute concentration of growth factors or the intensity of stimulation, we here introduce that some transient responses are prompted by temporal rate of increase of stimulation, rather than intensity of stimulation, by three independent mechanisms. These include the Ras system with fast activation and slow inactivation, the ERK-dependent negative feedback loop system, and the receptor degradation system, all of which can be commonly seen in various signaling networks. In addition, we show the distinct transient and steady state characteristics of these systems.     

The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady-state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4-10.9 times that of glutamine, and serine and branched-chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day(-1).     

The Light Growth Response of Phycomyces

With the help of an automated tracking system we have studied the characteristics of the transient light growth response of Phycomyces. The response shows a sharply defined latency. The Q₁₀ of the reciprocal latency is 2.4. Response patterns at different peaks of the action spectrum are the same. The gradual variation of response magnitude over a wide range of adapted intensifies parallels that of phototropism. The responses to saturating stimuli exhibit a strong oscillation with a constant period of 1.6 min and variable damping. The growth responses to sinusoidally varying light intensities show a system bandwidth of 2.5 x 10^-3 Hz. The linear dependence of phase shift on frequency is largely attributable to the latency observed with pulse stimuli. In the high intensity range a previously suspected increase of the steady-state growth rate with intensity has been confirmed. The light growth responses of mutants selected for diminished phototropism have been investigated. Many of these mutants have sizable but grossly distorted growth responses.     

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.     

Transient response simulations of recombinant microbial populations

An asynchronous bacterial population has been approximated using a finite number of "computer" cells, each based on a complex single-cell model for Escherichia coli. This formulation correctly simulates the transient responses of protein and total cell mass synthesis rate to the sudden increase in the concentration of limiting energy source in the growth medium. Experimentally observed responses of rRNA and mRNA synthesis rates to growth rate shifts are qualitatively mirrored by the model. Simulation trends following those of a rel(-) mutant suggest that model modifications are needed to describe the dynamics of the stringent response. Simulations of the responses of recombinant populations to plasmid amplification or plasmid promoter induction also result in behavior similar to that determined experimentally. The calculated responses for recombinant populations subjected to constant promoter induction or cyclic induction-noninduction lead to the conclusion that inducible systems give greater productivity than those with fixed promoter strength. This formulation may be utilized as a basis for exploring other aspects of recombinant population dynamics.     

Relative addition rate and external concentration; Driving variables used in plant nutrition research

Abstract An increasing literature accounting for various types of experiments indicates that far lower external nutrient concentrations are required by plants than is usually thought to be the case. It is concluded that the ion uptake capacity of the roots, as described by the carrier concept, is high compared to that required for maintenance of the internal concentration. Serious errors in experimental conclusions are associated with insufficient and constant nutrient addition rates. The main errors are caused by non-steady states of the plants both with regard to the internal nutrient concentrations and the relative growth rate. A dynamic concept has been proposed for direct use as the treatment variable within the range of sub-optimum nutrition. The nutritional factor is expressed as a flow, the relative nutrient addition rate in laboratory studies and the nutrient flux density in the field. The experimental use of the relative addition rate has led to steady-state nutrient status and relative growth rate and the interpretation of plant responses which differ fundamentally from accepted views. Thus, for instance, deficiency symptoms disappear, as in natural conditions, when the internal nitrogen concentration is stable, independent of level. The nutrition/growth relationships are very different from those observed when external concentration is varied. The regression line of relative growth rate on relative addition rate passes near to the origin at an angle close to 45 to the axes, which implies that the obtained relative growth rate approximates closely the treatment variable. A striking example of observed differences is the positive effect on nitrogen fixation exerted by high relative nitrogen addition rates compared to the well-known negative effect of increasing external nitrogen concentration. The application of fertilizer on the basis of the nutrient flux density concept provides the possibility of supplying fertilizers corresponding to the consumption potential of the vegetation and to the natural flux density resulting from mineralization in the soil. Nitrogen utilization is high under such conditions and the resulting feedback of nutrition on the mineralization rate suggests that there will be a long-term increase in fertility.     

An assessment of the role of physiological adaptation in the transient response of bacterial cultures

The RNA-limiting theory of transient response states that the primary physiological adaptation which occurs when microbial cultures are grown at specific rates less than their maximum is a decrease in the cellular level of RNA. It predicts that, as a result of this decrease, the response of the culture to a shift-up in growth rate will be limited by its RNA level. In order to test the RNA-limiting theory and to investigate the role physiological adaptation in transient response, experiments were performed in which steady-state chemostat cultures of Pseudomonasputida grown at various specific rates were transferred to batch reactors containing sufficient carbon source (L-lysine) and nutrients to remove all external growth restrictions. Samples were collected during the subsequent transient period for determination of the macromolecular composition and the maximum instantaneous oxygen uptake rate. The results indicated that, while decreases in the RNA level did significantly affect the nature of the transient response, other unidentified components varied with the steady-state specific growth rate at which the culture had been grown prior to the shift-up and that the levels of those components affected the nature of the subsequent transient response. This implies that the RNA-limiting theory is inadequate for describing the transient responses of cultures grown over a wide range of specific growth rates.     

The unsteady continuous culture of phosphate-limited Monochrysis lutheri droop: Experimental and theoretical analysis

A three-state variable model for phosphate-limited phytoplankton growth in a continuously lit continuous culture is proposed. In the model, the phosphate uptake rate per cell is a Michaelis-Mententype hyperbolic function of ambient nutrient concentration and the growth rate is a Droop-type hyperbolic function of cell quota. Steady-state and short-term uptake experiments with unialgal cultures of Monochrysis lutheri Droop, a marine chrysophyte, were used to calibrate the proposed model. For the long-term unsteady experiments, the model predicts well the culture's dynamic response in terms of cell density to steps down and up in influent concentration of limiting nutrient. For step changes in dilution rate, the model predicts well the culture's response to a step down but predicts poorly the culture's response to a step up. The long-term responses of the cultures to impulses in influent concentration show that the model fails to predict, even qualitatively, the behavior of the phytoplankton. Not unexpectedly, the model fails most dramatically in those experiments involving a rapid increase in cell quota, thereby demonstrating both the inherent flaws in the concept of the instantaneous growth rate as a function of instantaneous cell quota and the need for further dynamic characterization of phytoplankton behavior.     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Computer controlled transient state in a fed batch culture

Summary A controlled shift down of the specific growth rate in a fed batch culture is proposed for microbial product formation processes. The growth kinetics depend on three preset parameters: initial biomass concentration, initial specific growth rate, and the transient constant (time derivative of the specific growth rate). An experimental example is given.     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Transient response of continuous cultures to changes in temperature

The purpose of this experimental study was to examine the transient response of a chemostat-type continuous culture of Escherichia coli B to step changes in temperature by following transient limiting substrate concentration and calculating from it the transient growth rate. The transient response to step changes of temperature was tested for four different situations. In the first two cases, temperature was shifted down from 37 to 27°C., and 37 to 32°C. In the last two, it was shifted up from 32 to 37°C., and 27 to 37°C. When the temperature was shifted up, the growth rate increased rather rapidly to its transient maximum value and then decreased slowly until it, settled back into the steady-state value. On the other hand, when the temperature was shifted down, the growth rate decreased relatively rapidly to its transient minimum and then it slowly increased and returned gradually to the steady-state value. The magnitude of the transients was less than would be expected if the transient growth rates followed an Arrhenius function.     

Cell-mass maximization in fed-batch cultures

Complete solutions are provided for cell-mass maximization for free and fixed final times and constant and variable yields. The optimal feed rate profile is a concatenation of maximum, minimum and singular feed rates. The exact sequence and duration of each feed rate depends primarily on the initial substrate concentration, and degenerate cases arise due to the magnitude constraint on the feed rate and the length of final time t _f. When the final time is free and not in the performance index, it is infinite for constant yield so that any form of feed rate leads to the same amount of cells, while for variable yield the singular feed rate is exponential and maximizes the yield. For fixed final time the singular feed rate for constant yield is exponential and maximizes the specific growth rate by maintaining the substrate concentration constant, while for variable yield, it is semi-exponential and the substrate concentration starts near the maximum specific growth rate and moves toward the maximum yield. A simple sufficient condition for existence of singular feed rate requires an existence of a region bounded by the maxima of specific growth and cellular yield. Otherwise, the optimal feed rate profile is a bang-bang type and the bioreactor operates in batch mode.     

Study of the dynamic behavior of the chemostat system

This paper deals with a theoretical study on the dynamic, character of the chemostat system. It. is primarily based on the Monod model for growth limitation, although certain more complex models are considered. Since the Monod model is described in terms of two variables, an analysis by use of a phase plane plot will show the various possible types of behavior theoretically expected for transient conditions of the system. In this paper it will be shown that the chemostat system might show an overshoot (or an underswing) with respect to changes in cell and substrate concentrations, depending on the extent to which the system might be disturbed from steady-slate conditions. Other types of transient behavior ran also be expected when one of the system parameters such as dilution rate or input substrate concentration is disturbed in a stepwise manner. The simple Monod chemostat model was found never to oscillate in either a damped or a sustained manner as has been experimentally reported. Discussion is included about the transient behavior of other chemostat models such as that involving a variable yield coefficient, i.e., including the effect of cell maintenance requirements.     

Regulation of ribosome synthesis in Escherichia coli: effects of temperature and dilution rate changes

The effect of temperature on the synthesis of ribosome in Escherichia coli K-12 was investigated. In continuous fermentation, the total and functioning ribosome contents decreased with increasing temperature, while the non-functioning ribosome content remained unchanged. Cells contained higher amounts of functioning ribosome at lower temperatures to compensate for the decrease in translational activity. A transient study was performed to investigate the dynamic response of the cell to changes in the dilution rate. In response to the dilution rate shift-up, the cell mass decreased until the cells produced a sufficient amount of ribosomes to support the new higher growth rate. However, the response to the dilution rate shift-down resulted in an immediate increase in cell mass. This may be due to the fact that the cell already contains enough ribosomes to support a lower growth rate corresponding to the new low dilution rate. Based on the experimental results, a mathematical model was developed to describe the cell growth at transient as well as steady states. The total ribosome content was included as a variable because it affects the growth rate of the cell. (c) 1996 John Wiley & Sons, Inc.     

Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.     

The modelled growth of mycorrhizal and non-mycorrhizal plants under constant versus variable soil nutrient concentration

We studied the response of mycorrhizal and non-mycorrhizal plants to variation in soil nutrient concentration. A model for the relative growth rate (RGR) of plant biomass was constructed with soil nutrients as an explanatory variable. A literature survey was carried out to find the relative magnitudes of parameter values for mycorrhizal and non-mycorrhizal plants. Mycorrhizal plants had higher RGR at low nutrient concentrations and non-mycorrhizal plants at high nutrient concentrations. The RGR of mycorrhizal and non-mycorrhizal plants at constant versus log-normally distributed soil nutrient concentration were compared to see the effect of mycorrhizal status on responses to variation. Variation in nutrient concentration generally reduced RGR, especially in mycorrhizal plants. The RGR of a non-mycorrhizal plant may increase with variation where a growth function threshold exists, i.e. a soil nutrient concentration that must be exceeded to allow growth. Mycorrhizal plants appeared more sensitive to variation in nutrient concentration than non-mycorrhizal plants due to the higher affinity of mycorrhizal roots at low nutrient levels. However, this prediction may be reversed if mycorrhizal symbiosis considerably stabilises flow of nutrients to plant physiological processes, such that mycorrhizal plants experience less variation in soil nutrient concentration than non-mycorrhizal plants. Our results also attain broader significance by suggesting a general trade-off between competitive ability in a constant versus variable resource availability.     

INTERACTIONS AMONG PHOSPHATE UPTAKE,PHOTOSYNTHESIS, AND CHLOROPHYLL FLUORESCENCE IN NUTRIENT‐LIMITED CULTURES OF THE CHLOROPHYTE MICROALGA DUNALIELLA TERTIOLECTA1

Phosphate‐limited and phosphate‐sufficient continuous cultures of the marine chlorophyte microalga Dunaliella tertiolecta Butcher were examined for their responses to the addition of phosphate. Phosphate‐limited cultures showed a marked quenching of chl fluorescence following a pulse of phosphate. This response was absent from cells growing under phosphate‐sufficient conditions. Both the extent of fluorescence quenching (where present) and the initial rate of change in quenching were dependent on the concentration of phosphate added to cell suspensions and on the degree of limitation (growth rate in continuous culture). The addition of phosphate also brought about a transient decrease in photosynthetic oxygen evolution and a stimulation in respiration, which were relaxed as the added phosphate was depleted from the external medium. The applicability of using nutrient‐induced fluorescence transients as a tool to identify the nutrient status of phytoplankton populations is discussed.     

Ethylene emission and responsiveness to applied ethylene vary among Poa species that inherently differ in leaf elongation rates

A plant's ability to produce and respond to ethylene is essential for its vegetative growth. We studied whole-shoot ethylene emission and leaf growth responses to applied ethylene in four Poa spp. that differ inherently in leaf elongation rate and whole-plant relative growth rate. Compared with the fast-growing Poa annua and Poa trivialis, the shoots of the slow-growing species Poa alpina and Poa compressa emitted daily 30% to 50% less ethylene, and their leaf elongation rate was more strongly inhibited when ethylene concentration was increased up to 1 microL L(-1). To our surprise, however, low ethylene concentrations (0.02-0.03 microL L(-1)) promoted leaf growth in the two slow-growing species; at the same concentrations, leaf elongation rate of the two fast-growing species was only slightly inhibited. All responses were observed within 20 min after ethylene applications. Although ethylene generally inhibits growth, our results show that in some species, it may actually stimulate growth. Moreover, in the two slow-growing Poa spp., both growth stimulation and inhibition occurred in a narrow ethylene concentration range, and this effect was associated with a much lower ethylene emission. These findings suggest that the regulation of ethylene production rates and perception of the gas may be more crucial during leaf expansion of these species under non-stressful conditions and that endogenous ethylene concentrations are not large enough to saturate leaf growth responses. In the two fast-growing species, a comparatively higher ethylene endogenous concentration may conversely be present and sufficiently high to saturate leaf elongation responses, invariably leading to growth inhibition.