A basic set of homeostatic controller motifs.

Adaptation and homeostasis are essential properties of all living systems. However, our knowledge about the reaction kinetic mechanisms leading to robust homeostatic behavior in the presence of environmental perturbations is still poor. Here, we describe, and provide physiological examples of, a set of two-component controller motifs that show robust homeostasis. This basic set of controller motifs, which can be considered as complete, divides into two operational work modes, termed as inflow and outflow control. We show how controller combinations within a cell can integrate uptake and metabolization of a homeostatic controlled species and how pathways can be activated and lead to the formation of alternative products, as observed, for example, in the change of fermentation products by microorganisms when the supply of the carbon source is altered. The antagonistic character of hormonal control systems can be understood by a combination of inflow and outflow controllers.

Robust adaptation and homeostasis by autocatalysis.

Robust homeostatic mechanisms are essential for the protection and adaptation of organisms in a changing and challenging environment. Integral feedback is a control-engineering concept that leads to robust, i.e., perturbation-independent, adaptation and homeostatic behavior in the controlled variable. Addressing two-component negative feedback loops of a controlled variable A and a controller molecule E, we have shown that integral control is closely related to the presence of zero-order fluxes in the removal of the manipulated variable E. Here we show that autocatalysis is an alternative mechanism to obtain integral control. Although the conservative and marginal stability of the Lotka-Volterra oscillator (LVO) with autocatalysis in both A and E is often considered as a major inadequacy, homeostasis in the average concentrations of both A and E ( and ) is observed. Thus, autocatalysis does not only represent a mere driving force, but may also have regulatory roles.

Excision of uracil from DNA by the hyperthermophilic Afung protein is dependent on the opposite base and stimulated by heat-induced transition to a more open structure.

Hydrolytic deamination of DNA-cytosines into uracils is a major source of spontaneously induced mutations, and at elevated temperatures the rate of cytosine deamination is increased. Uracil lesions are repaired by the base excision repair pathway, which is initiated by a specific uracil DNA glycosylase enzyme (UDG). The hyperthermophilic archaeon Archaeoglobus fulgidus contains a recently characterized novel type of UDG (Afung), and in this paper we describe the over-expression of the afung gene and characterization of the encoded protein. Fluorescence and activity measurements following incubation at different temperatures may suggest the following model describing structure-activity relationships: At temperatures from 20 to 50 degrees C Afung exists as a compact protein exhibiting low enzyme activity, whereas at temperatures above 50 degrees C, the Afung conformation opens up, which is associated with the acquisition of high enzyme activity. The enzyme exhibits opposite base-dependent excision of uracil in the following order: U>U:T>U:C>>U:G>>U:A. Afung is product-inhibited by uracil and shows a pronounced inhibition by p-hydroxymercuribenzoate, indicating a cysteine residue essential for enzyme function. The Afung protein was estimated to be present in A. fulgidus at a concentration of approximately 1000 molecules per cell. Kinetic parameters determined for Afung suggest a significantly lower level of enzymatic uracil release in A. fulgidus as compared to the mesophilic Escherichia coli.

Biological timing and the clock metaphor: oscillatory and hourglass mechanisms.

Living organisms have developed a multitude of timing mechanisms--"biological clocks." Their mechanisms are based on either oscillations (oscillatory clocks) or unidirectional processes (hourglass clocks). Oscillatory clocks comprise circatidal, circalunidian, circadian, circalunar, and circannual oscillations--which keep time with environmental periodicities--as well as ultradian oscillations, ovarian cycles, and oscillations in development and in the brain, which keep time with biological timescales. These clocks mainly determine time points at specific phases of their oscillations. Hourglass clocks are predominantly found in development and aging and also in the brain. They determine time intervals (duration). More complex timing systems combine oscillatory and hourglass mechanisms, such as the case for cell cycle, sleep initiation, or brain clocks, whereas others combine external and internal periodicities (photoperiodism, seasonal reproduction). A definition of a biological clock may be derived from its control of functions external to its own processes and its use in determining temporal order (sequences of events) or durations. Biological and chemical oscillators are characterized by positive and negative feedback (or feedforward) mechanisms. During evolution, living organisms made use of the many existing oscillations for signal transmission, movement, and pump mechanisms, as well as for clocks. Some clocks, such as the circadian clock, that time with environmental periodicities are usually compensated (stabilized) against temperature, whereas other clocks, such as the cell cycle, that keep time with an organismic timescale are not compensated. This difference may be related to the predominance of negative feedback in the first class of clocks and a predominance of positive feedback (autocatalytic amplification) in the second class. The present knowledge of a compensated clock (the circadian oscillator) and an uncompensated clock (the cell cycle), as well as relevant models, are briefly re viewed. Hourglass clocks are based on linear or exponential unidirectional processes that trigger events mainly in the course of development and aging. An important hourglass mechanism within the aging process is the limitation of cell division capacity by the length of telomeres. The mechanism of this clock is briefly reviewed. In all clock mechanisms, thresholds at which "dependent variables" are triggered play an important role.

The Goodwin model: simulating the effect of light pulses on the circadian sporulation rhythm of Neurospora crassa.

The Goodwin oscillator is a minimal model that describes the oscillatory negative feedback regulation of a translated protein which inhibits its own transcription. Now, over 30 years later this scheme provides a basic description of the central components in the circadian oscillators of Neurospora, Drosophila, and mammals. We showed previously that Neurospora's resetting behavior by pulses of temperature, cycloheximide or heat shock can be simulated by this model, in which degradation processes play an important role for determining the clock's period and its temperature-compensation. Another important environmental factor for the synchronization is light. In this work, we show that on the basis of a light-induced transcription of the frequency (frq) gene phase response curves of light pulses as well as the influence of the light pulse length on phase shifts can be described by the Goodwin oscillator. A relaxation variant of the model predicts that directly after a light pulse inhibition in frq -transcription occurs, even when the inhibiting factor Z (FRQ) has not reached inhibitory concentrations. This has so far not been experimentally investigated for frq transcription, but it complies with a current model of light-induced transcription of other genes by a phosphorylated white-collar complex. During long light pulses, the relaxational model predicts that the sporulation rhythm is arrested in a steady state of high frq -mRNA levels. However, experimental results indicate the possibility of oscillations around this steady state and more in favor of the results by the original Goodwin model. In order to explain the resetting behavior by two light pulses, a biphasic first-order kinetics recovery period of the blue light receptor or of the light signal transduction pathway has to be assumed.

Heat shock and oxidative stress-induced exposure of hydrophobic protein domains as common signal in the induction of hsp68.

The hypothesis of a common signal for heat shock (HS) and oxidative stress (OS) was analyzed in C6 cells with regard to the induction of heat shock proteins (Hsps). The synthesis rate and level of the strictly inducible Hsp68 was significantly higher after HS (44 degrees C) compared with OS (2 mm H2O2). This difference corresponded to higher and lower activation of the heat shock factor (HSF) by HS and OS, respectively. OS, on the other hand, showed stronger cytotoxicity compared with HS as indicated by drastic lipid peroxidation and inhibition of protein synthesis as well as of mitochondrial and endocytotic activity. Lactic dehydrogenase also revealed stronger inhibition of enzyme activity by OS than by HS as shown in cells and in vitro experiments. Conformational analysis of lactic dehydrogenase by the fluorophore 1-anilinonaphtalene-8-sulfonic acid, however, showed stronger exposure of hydrophobic domains after HS than after OS which correlates positively with the Hsp68 response. Treatment of cells with deoxyspergualin, which exhibits high affinity to Hsps, the putative inhibitors of HSF, strongly increased only OS-induced hsp68 expression. In conclusion, the results suggest that exposure of hydrophobic domains of cytosolic proteins represents the common first signal in the multistep activation pathway of HSF.

pH homeostasis of the circadian sporulation rhythm in clock mutants of Neurospora crassa.

The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq+) and the mutants chr, frq1, frq7, and frq8. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4-9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq1. A loss of pH homeostasis of the period, however, was observed in the mutants frq7 and frq8, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H2PO4- ion has been found, indicating that the uptake of H2PO4- may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq7 and frq8 mutants may be related to less easily degradable FRQ7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ7 protein degradation increases and should lead to shorter period lengths.

Hysteretic enzyme adaptation to environmental pH: change in storage pH of alkaline phosphatase leads to a pH-optimum in the opposite direction to the applied change.

The activity of alkaline phosphatase (AP) shows a change in optimum pH in the opposite direction to the applied change in storage pH. Typically, a change in storage pH from 9.8 to 8.5 results in a (reversible) change of the pH-optimum from 10.0 to 10.8. Protein fluorescence analysis shows that this response is probably due to conformational changes induced by the different storage conditions. As storage pH increases, a more 'open' or less 'compact' conformation is attained. Analysis of the diprotic model (a model which describes possible pH-responses of enzymes) indicates, that, as the AP conformation is getting more 'open' an increase in the dissociation of activity-regulating protons of AP occurs. This leads to a decrease in pH-optimum, precisely as found in the experiment. The prerequisite for such a response, however, is that the conformational adaptation to environmental assay pH is slow (hysteretic) when compared with assay time (400 s). The relaxation time of this adaptation was found to be in the order of 2 h.

The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa.

The Goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of Neurospora and Drosophila. An essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. When protein synthesis in N. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (CHX) the period of the circadian sporulation rhythmicity is only slightly increased. The explanation of this effect may be seen in the inhibition of protein synthesis and protein degradation. In the model, increasing inhibition of both processes led to very similar results with respect to period length. That protein degradation is, in fact, inhibited by CHX is shown by determining protein degradation in N. crassa by means of pulse chase experiments. Phase response curves (PRCs) of the N. crassa sporulation rhythm toward CHX which were reported in the literature and investigated in this paper revealed significant differences between frq+and the long period mutants frq7and csp -1 frq7. These PRCs were also convincingly simulated by the model, if a transient inhibition of protein degradation by CHX is assumed as well as a lower constitutive degradation rate of FRQ-protein in the frq7/ csp -1 frq7mutants. The lower sensitivities of frq7and csp -1 frq7towards CHX may thus be explained by a lower degradation rate of clock protein FRQ7. The phase shifting by moderate temperature pulses (from 25 to 30 degrees C) can also be simulated by the Goodwin model and shows large phase advances at about CT 16-20 as observed in experiments. In case of higher temperature pulses (from 35 to 42 or 45 degrees C=heat shock) the phase position and form of the PRC changes as protein synthesis is increasingly inhibited. It is known from earlier experiments that heat shock not only inhibits the synthesis of many proteins but also inhibits protein degradation. Taking this into account, the Goodwin model also simulates the PRCs of high temperature (heat shock) pulses.

The Goodwin oscillator: on the importance of degradation reactions in the circadian clock.

This article focuses on the Goodwin oscillator and related minimal models, which describe negative feedback schemes that are of relevance for the circadian rhythms in Neurospora, Drosophila, and probably also in mammals. The temperature behavior of clock mutants in Neurospora crassa and Drosophila melanogaster are well described by the Goodwin model, at least on a semi-quantitative level. A similar semi-quantitative description has been found for Neurospora crassa phase response curves with respect to moderate temperature pulses, heat shock pulses, and pulses of cycloheximide. A characteristic feature in the Goodwin and related models is that degradation of clock-mRNA and clock protein species plays an important role in the control of the oscillator's period. As predicted by this feature, recent experimental results from Neurospora crassa indicate that the clock (FRQ) protein of the long period mutant frq7 is degraded approximately twice as slow as the corresponding wild-type protein. Quantitative RT-PCR indicates that experimental frq7-mRNA concentrations are significantly higher than wild-type levels. The latter findings cannot be modeled by the Goodwin oscillator. Therefore, a threshold inhibition mechanism of transcription is proposed.

Temperature adaptation of house keeping and heat shock gene expression in Neurospora crassa.

Adaptation of house keeping and heat shock gene expression was determined in Neurospora crassa during continuous exposure to different temperatures. Steady-state values of total protein synthesis differed little after incubation for 24 h at temperatures between 15 and 42 degreesC. Adaptation kinetics at 42 degreesC showed an initial, transient inhibition of total protein synthesis. Similar kinetics were observed with actin synthesis and tubulin mRNA. A priming 1-h heat shock of 42 degreesC 2 h prior to a second continuous exposure to 42 degreesC abolished the inhibitory effect of the second treatment and resulted in "acquired translational tolerance." Steady-state values of HSP70 synthesis rates revealed increasing levels with increasing temperatures after incubation for 24 h at different temperatures. Adaptation kinetics of the synthesis rates of different HSPs in vivo revealed maximal rates after 2 h and then a decrease to the elevated steady-state levels. The total amount of the major constitutive and inducible HSP70 isoform as determined by Western blots reached a maximum 2 h after the beginning of 42 degreesC exposure and only a slight decrease (25%) of the maximal value after 24 h. The inducible isoform of HSP70, in contrast, reached a maximum after 4-8 h and then decreased strongly after 24 h. HSP mRNAs reached maximal amounts 45-60 min after the beginning of 42 degreesC exposure and then declined after 8 h as determined by in vitro translation. Northern blots revealed maximal mRNA amounts of the inducible HSP70 after 30 min and zero amounts after 4 h exposure to 42 degreesC. After a shift to 42 degreesC HSP70 isoforms were immediately translocated into the nucleus and reshuttled into the cytoplasm during the following 6 h. The nuclear content of HSP70 remained elevated during the adapted steady state at 24 h. It is concluded that the adapted state after 24 h is based on enhanced amounts of constitutive isoforms in the cytoplasm and in the nucleus, whereas the inducible isoforms of HSP70 show faster adaptation kinetics.

Temperature compensation of the circadian period length--a special case among general homeostatic mechanisms of gene expression?

In Neurospora crassa, as well as in other organisms, the expression of housekeeping genes is transiently suppressed after exposure to higher temperatures (30-45 degrees C); expression is then reactivated and adapts after a few-hours to values closer to the initial rates. Adaptive mechanisms apparently exist in the processes of transcription, RNA processing, and translation and render protein synthesis rates temperature compensated. Heat shock proteins (HSPs) play an important role within these mechanisms ("acquired thermotolerance of protein synthesis"), but their function is as yet not exactly known. Adaptive mechanisms seem also to involve intracellular ion changes after exposure to moderate temperature elevation. The expression of heat shock genes is transiently enhanced after exposure to higher temperatures and also adapts after a few hours. The adaptation mechanism includes inactivation of the heat shock transcription factor (HSF) by means of phosphorylation changes and possibly by binding of a gene product (HSP70)-a mechanism representing a negative feedback control. These examples demonstrate the existence of general adaptive mechanisms at different levels of gene expression that may also be at work in the temperature compensation of clock gene expression. Apart from such adaptation processes, antagonistic reactions within the processes of gene expression and protein modification might be equally enhanced or suppressed by temperature changes, leaving the equilibrium unaffected or balanced (antagonistic balance, see Ruoff et al., this issue of Chronobiology International). This principle is shown to apply to the effect of temperature elevation on total protein synthesis and degradation. It may also apply to other antagonistic processes such as phosphorylation-dephosphorylation or monomer-dimer formation. The circadian clock mechanism is assumed to consist of several processes that can either adapt or produce a balance. Single amino acid changes in a clock protein are assumed to partially upset this adaptation or balance.

Modeling temperature compensation in chemical and biological oscillators.

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of "antagonistic balance" has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths.

Characterization of Nitrate Reductase from Light- and Dark-Exposed Leaves (Comparison of Different Species and Effects of 14-3-3 Inhibitor Proteins).

Nitrate reductase (NR) was extracted and partially purified from leaves of squash (Curcurbita maxima), spinach (Spinacia oleracea), and three transgenic Nicotiana plumbaginifolia leaves in the presence of phosphatase inhibitors to preserve its phosphorylation state. Purified squash NR showed activation by substrates (hysteresis) when prepared from leaves in the light as well as in darkness. A 14-3-3 protein known to inhibit phosphorylated spinach NR in the presence of Mg2+ decreased by 70 to 85% the activity of purified NR from dark-exposed leaves, whereas NR from light-exposed leaves decreased by 10 to 25%. Apparent lack of posttranslational NR regulation in a transgenic N. plumbaginifolia expressing an NR construct with an N-terminal deletion ([delta]NR) may be explained by more easy dissociation of 14-3-3 proteins from [delta]NR. Partially purified [delta]NR was, however, inhibited by 14-3-3 protein, and the binding constant of 14-3-3 protein (4 x 108 M-1) and the NR-inhibiting protein concentration that results in a 50% reduction of free NR (2.5 nM) were the same for NR and [delta]NR. Regulation of NR activity by phosphorylation and binding of 14-3-3 protein was a general feature for all plants tested, whereas activation by substrates as a possible regulation mechanism was verified only for squash.

Evidence for increased proton dissociation in low-activity forms of dephosphorylated squash-leaf nitrate reductase.

The pH dependence of squash-leaf nitrate reductase has been studied. It has been found that high- and low-activity forms of purified nitrate reductase (both forms dephosphorylated) have different optimum pH values. A high-activity form has always a higher pH optimum compared with a low-activity form. Model computations show that the decrease in activity and the corresponding change of the pH optimum is apparently due to a conformation-dependent increase of proton dissociation of the enzyme. As previously shown, this behavior is also observed in leaf extracts during the conversion (and probably phosphorylation of nitrate reductase) from a high-active form to a low-active form when plants are transferred from light to darkness.

Hysteretic behavior of nitrate reductase. Evidence of an allosteric binding site for reduced pyridine nucleotides.

In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.

Molecular oxygen as electron acceptor in the NADH-nitrate reductase system.

This paper describes first experimental evidence that dissolved molecular oxygen acts as an electron acceptor in the NADH-nitrate reductase system. The molecular mechanism and possible physiological implications on the induction mechanism of nitrate reductase by nitrate ion are discussed.