Excision of uracil from DNA by hSMUG1 includes strand incision and processing.

Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3'-α,ß-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5'-phosphate. UIP is removed from the 3'-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3'-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2' occurs via the formation of a C1' enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.

The Goodwin Oscillator and its Legacy.

In the 1960's Brian Goodwin published a couple of mathematical models showing how feedback inhibition can lead to oscillations and discussed possible implications of this behaviour for the physiology of the cell. He also presented key ideas about the rich dynamics that may result from the coupling between such biochemical oscillators. Goodwin's work motivated a series of theoretical investigations aiming at identifying minimal mechanisms to generate limit cycle oscillations and deciphering design principles of biological oscillators. The three-variable Goodwin model (adapted by Griffith) can be seen as a core model for a large class of biological systems, ranging from ultradian to circadian clocks. We summarize here main ideas and results brought by Goodwin and review a couple of modeling works directly or indirectly inspired by Goodwin's findings.

DOPA Homeostasis by Dopamine: A Control-Theoretic View.

Dopamine (DA) is an important signal mediator in the brain as well as in the periphery. The term "dopamine homeostasis" occasionally found in the literature refers to the fact that abnormal DA levels can be associated with a variety of neuropsychiatric disorders. An analysis of the negative feedback inhibition of tyrosine hydroxylase (TH) by DA indicates, with support from the experimental data, that the TH-DA negative feedback loop has developed to exhibit 3,4-dihydroxyphenylalanine (DOPA) homeostasis by using DA as a derepression regulator. DA levels generally decline when DOPA is removed, for example, by increased oxidative stress. Robust DOPA regulation by DA further implies that maximum vesicular DA levels are established, which appear necessary for a reliable translation of neural activity into a corresponding chemical transmitter signal. An uncontrolled continuous rise (windup) in DA occurs when Levodopa treatment exceeds a critical dose. Increased oxidative stress leads to the successive breakdown of DOPA homeostasis and to a corresponding reduction in DA levels. To keep DOPA regulation robust, the vesicular DA loading requires close to zero-order kinetics combined with a sufficiently high compensatory flux provided by TH. The protection of DOPA and DA due to a channeling complex is discussed.

Considerably Lower Levels of Hypocretin-1 in Cerebrospinal Fluid Is Revealed by a Novel Mass Spectrometry Method Compared with Standard Radioimmunoassay.

Low levels of hypocretin-1 (Hcrt1) in cerebrospinal fluid (CSF) are associated with narcolepsy type 1 (NT1). Although immunoassays are prone to antibody batch differences, detection methods and variation between laboratories, the standard method for Hcrt1 measurement is a radioimmunoassay (RIA). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an antibody- and radioactive free alternative for precise measurement of Hcrt1. We developed an LC-MS/MS method for measurement of Hcrt1 in CSF with automated sample preparation by solid-phase extraction (SPE). The LC-MS/MS method was compared with the RIA method for Hcrt1 detection. CSF samples from healthy subjects and NT1 patients was obtained by lumbar puncture. NT1 patients were diagnosed according to the minimal criteria by the International Classification of Sleep Disorders (ICSD). The LC-MS/MS method showed linearity across the range of calibrators and had a limit of detection (LOD) of 2.5 pg/mL and a limit of quantitation (LOQ) of 3.6 pg/mL. Comparison of the LC-MS/MS method with RIA revealed a 19 times lower level in healthy controls and 22 times lower level in NT1 patients with the LC-MS/MS method than with RIA. Bland-Altman analysis demonstrated agreement between the methods. These results question what is detected by RIA and strongly suggest that the physiological concentrations of the peptide are much lower than previously believed. LC-MS/MS proves to be an alternative for detection of Hcrt1 for diagnosis of narcolepsy.

Interleukin-1-related activity and hypocretin-1 in cerebrospinal fluid contribute to fatigue in primary Sjögren's syndrome.

BACKGROUND: Fatigue is a common and sometimes debilitating phenomenon in primary Sjögren's syndrome (pSS) and other chronic inflammatory diseases. We aimed to investigate how IL-1 ß-related molecules and the neuropeptide hypocretin-1 (Hcrt1), a regulator of wakefulness, influence fatigue. METHODS: Hcrt1 was measured by radioimmunoassay (RIA) in cerebrospinal fluid (CSF) from 49 patients with pSS. Interleukin-1 receptor antagonist (IL-1Ra), IL-1 receptor type 2 (IL-1RII), IL-6, and S100B protein were measured by enzyme-linked immunosorbent assay (ELISA). Fatigue was rated by the fatigue visual analog scale (fVAS). RESULTS: Simple univariate regression and multiple regression analyses with fatigue as a dependent variable revealed that depression, pain, and the biochemical variable IL-1Ra had a significant association with fatigue. In PCA, two significant components were revealed. The first component (PC1) was dominated by variables related to IL-1ß activity (IL-1Ra, IL-1RII, and S100B). PC2 showed a negative association between IL-6 and Hcrt1. fVAS was then introduced as an additional variable. This new model demonstrated that fatigue had a higher association with the IL-1ß-related PC1 than to PC2. Additionally, a third component (PC3) became significant between low Hcrt1 concentrations and fVAS scores. CONCLUSIONS: The main findings of this study indicate a functional network in which several IL-1ß-related molecules in CSF influence fatigue in addition to the classical clinical factors of depression and pain. The neuropeptide Hcrt1 seems to participate in fatigue generation, but likely not through the IL-1 pathway.

Sensitivity analysis of a pathway with respect to fast and small temperature change.

Temperature affects all enzymes simultaneously in a metabolic system. The enzyme concentration in a biochemical system can be considered as invariant under fast and small temperature change. Therefore, the total sensitivity of a steady state flux through a pathway with respect to the temperature can be expressed as: the apparent activation energy of a steady state pathway flux equals the sum of weighted activation energies of the individual reactions contributing to the flux, where the weighting factors are the flux control coefficients of these reactions in the context of the network. Correspondingly, since the elasticity of any enzyme with respect to temperature is always nonzero, only the reactions with a nonzero flux control coefficient contribute accordingly to the temperature sensitivity of the pathway.

Modeling the diversion of primary carbon flux into secondary metabolism under variable nitrate and light/dark conditions.

In plants, the partitioning of carbon resources between growth and defense is detrimental for their development. From a metabolic viewpoint, growth is mainly related to primary metabolism including protein, amino acid and lipid synthesis, whereas defense is based notably on the biosynthesis of a myriad of secondary metabolites. Environmental factors, such as nitrate fertilization, impact the partitioning of carbon resources between growth and defense. Indeed, experimental data showed that a shortage in the nitrate fertilization resulted in a reduction of the plant growth, whereas some secondary metabolites involved in plant defense, such as phenolic compounds, accumulated. Interestingly, sucrose, a key molecule involved in the transport and partitioning of carbon resources, appeared to be under homeostatic control. Based on the inflow/outflow properties of sucrose homeostatic regulation we propose a global model on how the diversion of the primary carbon flux into the secondary phenolic pathways occurs at low nitrate concentrations. The model can account for the accumulation of starch during the light phase and the sucrose remobilization by starch degradation during the night. Day-length sensing mechanisms for variable light-dark regimes are discussed, showing that growth is proportional to the length of the light phase. The model can describe the complete starch consumption during the night for plants adapted to a certain light/dark regime when grown on sufficient nitrate and can account for an increased accumulation of starch observed under nitrate limitation.

A rapid method for preparation of the cerebrospinal fluid proteome.

The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high-abundance proteins (HAPs) can interfere with the detection of low-abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects.

Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model.

The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na(+) and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na(+)/K(+) homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis.

Background compensation revisited: Conserved phase response curves in frequency controlled homeostats with coherent feedback.

Background compensation is the ability of a controlled variable to respond to an applied perturbation in an unchanged manner and independent of different but constant background signals which act in parallel to the perturbation. Background compensation occurs by 'coherent feedback' mechanisms where additional control variables feed directly back to the controlled variable. This paper extends a previous study on background compensation to include phase responses in frequency controlled coherent feedback oscillators. While the frequency resetting amplitude in coherent feedback oscillators is found to be dependent on the inflow/outflow perturbation of the controlled variable and thereby become phase dependent, the frequency resetting itself and the corresponding phase response curves are found to be background compensated. It is speculated that this type of background compensation may be an additional way how ambient noise can be 'ignored' by organisms.

Coherent feedback leads to robust background compensation in oscillatory and non-oscillatory homeostats.

When in a reaction kinetic integral controller a step perturbation is applied besides a constant background, the concentration of a controlled variable (described as A) will generally respond with decreased response amplitudes ΔA as backgrounds increase. The controller variable E will at the same time provide the necessary compensatory flux to move A back to its set-point. A typical example of decreased response amplitudes at increased backgrounds is found in retinal light adaptation. Due to remarks in the literature that retinal light adaptation would also involve a compensation of backgrounds we became interested in conditions how background compensation could occur. In this paper we describe novel findings how background influences can be robustly eliminated. When such a background compensation is active, oscillatory controllers will respond to a defined perturbation with always the same (damped or undamped) frequency profile, or in the non-oscillatory case, with the same response amplitude ΔA, irrespective of the background level. To achieve background compensation we found that two conditions need to apply: (i) an additional set of integral controllers (here described as I1 and I2) have to be employed to keep the manipulated variable E at a defined set-point, and (ii), I1 and I2 need to feed back to the A-E signaling axis directly through the controlled variable A. In analogy to a similar feedback applied in quantum control theory, we term these feedback conditions as 'coherent feedback'. When analyzing retinal light adaptations in more detail, we find no evidence of the presence of background compensation mechanisms. Although robust background compensation, as described theoretically here, appears to be an interesting regulatory property, relevant biological or biochemical examples still need to be identified.

Homeostasis at different backgrounds: The roles of overlayed feedback structures in vertebrate photoadaptation.

We have studied the resetting behavior of eight basic integral controller motifs with respect to different but constant backgrounds. We found that the controllers split symmetrically into two classes: one class, based on derepression of the compensatory flux, leads to more rapid resetting kinetics as backgrounds increase. The other class, which directly activates the compensatory flux, shows a slowing down in the resetting at increased backgrounds. We found a striking analogy between the resetting kinetics of vertebrate photoreceptors and controllers based on derepression, i.e. vertebrate rod or cone cells show decreased sensitivities and accelerated response kinetics as background illuminations increase. The central molecular model of vertebrate photoadaptation consists of an overlay of three negative feedback loops with cytosolic calcium ([Formula: see text]), cyclic guanosine monophosphate (cGMP) and cyclic nucleotide-gated (CNG) channels as components. While in one of the feedback loops the extrusion of [Formula: see text] by potassium-dependent sodium-calcium exchangers (NCKX) can lead to integral control with cGMP as the controlled variable, the expected robust perfect adaptation of cGMP is lost, because of the two other feedback loops. They avoid that [Formula: see text] levels become too high and toxic. Looking at psychophysical laws, we found that in all of the above mentioned basic controllers Weber's law is followed when a "just noticeable difference" (threshold) of 1% of the controlled variable's set-point was considered. Applying comparable threshold pulses or steps to the photoadaptation model we find, in agreement with experimental results, that Weber's law is followed for relatively high backgrounds, while Stephens' power law gives a better description when backgrounds are low. Limitations of our photoadaption model, in particular with respect to potassium/sodium homeostasis, are discussed. Finally, we discuss possible implication of background perturbations in biological controllers when compensatory fluxes are based on activation.

Modeling the interaction between donor-derived regulatory T cells and effector T cells early after allogeneic hematopoietic stem cell transplantation.

While allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative therapy against hematological malignancies, modulation of donor T cell alloreactivity is required to enhance the graft-versus-leukemia (GVL) effect and control graft-versus-host-disease (GVHD) after allo-HSCT. Donor-derived regulatory CD4+CD25+Foxp3+ T cells (Tregs) play a central role in establishing of immune tolerance after allo-HSCT. They could be a key target to be modulated for increasing the GVL effect and control of GVHD. We constructed an ordinary differential equation model incorporating bidirectional interactions between Tregs and effector CD4+ T cells (Teffs) as a mechanism for control of Treg cell concentration. The goal is to elucidate how the interaction between Tregs and Teffs is modulated in order to get insights into fine tuning of alloreactivity after allo-HSCT. The model was calibrated with respect to published Treg and Teff recovery data after allo-HSCT. The calibrated model exhibits perfect or near-perfect adaptation to stepwise perturbations between Treg and Teff interactions, as seen in Treg cell populations when patients with relapsed malignancy were treated with anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4). In addition, the model predicts observed shifts of Tregs and Teffs concentrations after co-stimulatory receptor IL-2R or TNFR2 blockade with allo-HSCT. The present results suggest simultaneous blockades of co-stimulatory and co-inhibitory receptors as a potential treatment for enhancing the GVL effect after allo-HSCT without developing GVHD.

Integrating fluctuating nitrate uptake and assimilation to robust homeostasis.

Nitrate is an important nitrogen source used by plants. Despite of the considerable variation in the amount of soil nitrate, plants keep cytosolic nitrate at a homeostatic controlled level. Here we describe a set of homeostatic controller motifs and their interaction that can maintain robust cytosolic nitrate homeostasis at fluctuating external nitrate concentrations and nitrate assimilation levels. The controller motifs are divided into two functional classes termed as inflow and outflow controllers. In the presence of high amounts of environmental nitrate, the function of outflow controllers is associated to efflux mechanisms removing excess of nitrate from the cytosol that is taken up by low-affinity transporter systems (LATS). Inflow controllers on the other hand maintain homeostasis in the presence of a high demand of nitrate by the cell relative to the amount of available environmental nitrate. This is achieved by either remobilizing nitrate from a vacuolar store, or by taking up nitrate by means of high-affinity transporter systems (HATS). By combining inflow and outflow controllers we demonstrate how nitrate uptake, assimilation, storage and efflux are integrated to a regulatory network that maintains cytosolic nitrate homeostasis at changing environmental conditions.

Kinetics and mechanisms of catalyzed dual-E (antithetic) controllers.

Homeostasis plays a central role in our understanding how cells and organisms are able to oppose environmental disturbances and thereby maintain an internal stability. During the last two decades there has been an increased interest in using control engineering methods, especially integral control, in the analysis and design of homeostatic networks. Several reaction kinetic mechanisms have been discovered which lead to integral control. In two of them integral control is achieved, either by the removal of a single control species E by zero-order kinetics ("single-E controllers"), or by the removal of two control species by second-order kinetics ("antithetic or dual-E control"). In this paper we show results when the control species E1 and E2 in antithetic control are removed enzymatically by ping-pong or ternary-complex mechanisms. Our findings show that enzyme-catalyzed dual-E controllers can work in two control modes. In one mode, one of the two control species is active, but requires zero-order kinetics in its removal. In the other mode, both controller species are active and both are removed enzymatically. Conditions for the two control modes are put forward and biochemical examples with the structure of enzyme-catalyzed dual-E controllers are discussed.

An amplified derepression controller with multisite inhibition and positive feedback.

How organisms are able to maintain robust homeostasis has in recent years received increased attention by the use of combined control engineering and kinetic concepts, which led to the discovery of robust controller motifs. While these motifs employ kinetic conditions showing integral feedback and homeostasis for step-wise perturbations, the motifs' performance differ significantly when exposing them to time dependent perturbations. One type of controller motifs which are able to handle exponentially and even hyperbolically growing perturbations are based on derepression. In these controllers the compensatory reaction, which neutralizes the perturbation, is derepressed, i.e. its reaction rate is increased by the decrease of an inhibitor acting on the compensatory flux. While controllers in this category can deal well with different time-dependent perturbations they have the disadvantage that they break down once the concentration of the regulatory inhibitor becomes too low and the compensatory flux has gained its maximum value. We wondered whether it would be possible to bypass this restriction, while still keeping the advantages of derepression kinetics. In this paper we show how the inclusion of multisite inhibition and the presence of positive feedback loops lead to an amplified controller which is still based on derepression kinetics but without showing the breakdown due to low inhibitor concentrations. By searching for the amplified feedback motif in natural systems, we found it as a part of the plant circadian clock where it is highly interlocked with other feedback loops.

Alleviation of C⋠C Mismatches in DNA by the Escherichia coli Fpg Protein.

DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The k cat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The K M values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N 4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

Personalized Medicine to Improve Treatment of Dopa-Responsive Dystonia-A Focus on Tyrosine Hydroxylase Deficiency.

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients.

Harmonic oscillations in homeostatic controllers: Dynamics of the p53 regulatory system.

Homeostatic mechanisms are essential for the protection and adaptation of organisms in a changing and challenging environment. Previously, we have described molecular mechanisms that lead to robust homeostasis/adaptation under inflow or outflow perturbations. Here we report that harmonic oscillations occur in models of such homeostatic controllers and that a close relationship exists between the control of the p53/Mdm2 system and that of a homeostatic inflow controller. This homeostatic control model of the p53 system provides an explanation why large fluctuations in the amplitude of p53/Mdm2 oscillations may arise as part of the homeostatic regulation of p53 by Mdm2 under DNA-damaging conditions. In the presence of DNA damage p53 is upregulated, but is subject to a tight control by Mdm2 and other factors to avoid a premature apoptotic response of the cell at low DNA damage levels. One of the regulatory steps is the Mdm2-mediated degradation of p53 by the proteasome. Oscillations in the p53/Mdm2 system are considered to be part of a mechanism by which a cell decides between cell cycle arrest/DNA repair and apoptosis. In the homeostatic inflow control model, harmonic oscillations in p53/Mdm2 levels arise when the binding strength of p53 to degradation complexes increases. Due to the harmonic character of the oscillations rapid fluctuating noise can lead, as experimentally observed, to large variations in the amplitude of the oscillation but not in their period, a behavior which has been difficult to simulate by deterministic limit-cycle models. In conclusion, the oscillatory response of homeostatic controllers may provide new insights into the origin and role of oscillations observed in homeostatically controlled molecular networks.

Frequency switching between oscillatory homeostats and the regulation of p53.

Homeostasis is an essential concept to understand the stability of organisms and their adaptive behaviors when coping with external and internal assaults. Many hormones that take part in homeostatic control come in antagonistic pairs, such as glucagon and insulin reflecting the inflow and outflow compensatory mechanisms to control a certain internal variable, such as blood sugar levels. By including negative feedback loops homeostatic controllers can exhibit oscillations with characteristic frequencies. In this paper we demonstrate the associated frequency changes in homeostatic systems when individual controllers -in a set of interlocked feedback loops- gain control in response to environmental changes. Taking p53 as an example, we show how Per2, ATM and Mdm2 feedback loops -interlocked with p53- gain individual control in dependence to the level of DNA damage, and how each of these controllers provide certain functionalities in their regulation of p53. In unstressed cells, the circadian regulator Per2 ensures a basic p53 level to allow its rapid up-regulation in case of DNA damage. When DNA damage occurs the ATM controller increases the level of p53 and defends it towards uncontrolled degradation, which despite DNA damage, would drive p53 to lower values and p53 dysfunction. Mdm2 on its side keeps p53 at a high but sub-apoptotic level to avoid premature apoptosis. However, with on-going DNA damage the Mdm2 set-point is increased by HSP90 and other p53 stabilizers leading finally to apoptosis. An emergent aspect of p53 upregulation during cell stress is the coordinated inhibition of ubiquitin-independent and ubiquitin-dependent degradation reactions. Whether oscillations serve a function or are merely a by-product of the controllers are discussed in view of the finding that homeostatic control of p53, as indicated above, does in principle not require oscillatory homeostats.