Ludger Rensing

Ethical Principles and Standards for the Conduct of Human and Animal Biological Rhythm Research

Most research papers published in Chronobiology International report the findings of investigations conducted on laboratory animals and human beings. The Journal, its editors and the publication committee endorse the compliance of investigators to the principles of the Declaration of Helsinki of the World Medical Association relating to the conduct of ethical research on human beings and the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Research of the National Research Council relating to the conduct of ethical research on laboratory and other animals. Chronobiology International requires that submitted manuscripts reporting the findings of human and animal research conform to the respective policy and mandates of the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals. The peer review of manuscripts will thus include judgment of whether or not the involved research methods conform to the standards of good research practice. This article outlines the basic expectations for the methods of human and animal biological rhythm research, both from the perspective of the fundamental criteria necessary for quality chronobiology investigation and from the perspective of humane and ethical research on human beings and animals.

TEMPERATURE EFFECT ON ENTRAINMENT, PHASE SHIFTING, AND AMPLITUDE OF CIRCADIAN CLOCKS AND ITS MOLECULAR BASES

Effects of temperature and temperature changes on circadian clocks in cyanobacteria, unicellular algae, and plants, as well as fungi, arthropods, and vertebrates are reviewed. Periodic temperature with periods around 24 h even in the low range of 1–2°C (strong Zeitgeber effect) can entrain all ectothermic (poikilothermic) organisms. This is also reflected by the phase shifts—recorded by phase response curves (PRCs)—that are elicited by step- or pulsewise changes in the temperature. The amount of phase shift (weak or strong type of PRC) depends on the amplitude of the temperature change and on its duration when applied as a pulse. Form and position of the PRC to temperature pulses are similar to those of the PRC to light pulses. A combined high/low temperature and light/dark cycle leads to a stabile phase and maximal amplitude of the circadian rhythm—when applied in phase (i.e., warm/light and cold/dark). When the two Zeitgeber cycles are phase-shifted against each other the phase of the circadian rhythm is determined by either Zeitgeber or by both, depending on the relative strength (amplitude) of both Zeitgeber signals and the sensitivity of the species/individual toward them. A phase jump of the circadian rhythm has been observed in several organisms at a certain phase relationship of the two Zeitgeber cycles. Ectothermic organisms show inter- and intraspecies plus seasonal variations in the temperature limits for the expression of the clock, either of the basic molecular mechanism, and/or the dependent variables. A step-down from higher temperatures or a step-up from lower temperatures to moderate temperatures often results in initiation of oscillations from phase positions that are about 180° different. This may be explained by holding the clock at different phase positions (maximum or minimum of a clock component) or by significantly different levels of clock components at the higher or lower temperatures. Different permissive temperatures result in different circadian amplitudes, that usually show a species-specific optimum. In endothermic (homeothermic) organisms periodic temperature changes of about 24 h often cause entrainment, although with considerable individual differences, only if they are of rather high amplitudes (weak Zeitgeber effects). The same applies to the phase-shifting effects of temperature pulses. Isolated bird pineals and rat suprachiasmatic nuclei tissues on the other hand, respond to medium high temperature pulses and reveal PRCs similar to that of light signals. Therefore, one may speculate that the self-selected circadian rhythm of body temperature in reptiles or the endogenously controlled body temperature in homeotherms (some of which show temperature differences of more than 2°C) may, in itself, serve as an internal entraining system. The so-called heterothermic mammals (undergoing low body temperature states in a daily or seasonal pattern) may be more sensitive to temperature changes. Effects of temperature elevation on the molecular clock mechanisms have been shown in Neurospora (induction of the frequency (FRQ) protein) and in Drosophila (degradation of the period (PER) and timeless (TIM) protein) and can explain observed phase shifts of rhythms in conidiation and locomotor activity, respectively. Temperature changes probably act directly on all processes of the clock mechanism some being more sensitive than the others. Temperature changes affect membrane properties, ion homeostasis, calcium influx, and other signal cascades (cAMP, cGMP, and the protein kinases A and C) (indirect effects) and may thus influence, in particular, protein phosphorylation processes of the clock mechanism. The temperature effects resemble to some degree those induced by light or by light-transducing neurons and their transmitters. In ectothermic vertebrates temperature changes significantly affect the melatonin rhythm, which in turn exerts entraining (phase shifting) functions.

The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast.

In S. cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of [31P]NMR spectroscopy. The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM]. Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable. HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock. The synthesis rates then decline. HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C. The similar dose dependencies of HSP synthesis and cytoplasmic pH. as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S. cerevisiae (Q0). This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation. No correlation was observed between HSP synthesis rate and total intracellular ATP content.

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 oscillators period. As predicted by this feature, recent experimental results from Neurospora crassa indicate that the clock (FRQ) protein of the long period mutant frq 7 is degraded approximately twice as slow as the corresponding wild-type protein. Quantitative RT-PCR indicates that experimental frq 7 -mRNA concentrations are significantly higher than wild-type levels. The latter findings cannot be modeled by the Goodwin oscillator.Therefore,athresholdinhibitionmechanismoftranscriptionis proposed.

Similar dose response of heat shock protein synthesis and intracellular pH change in yeast

In Saccharomyces cerevisiae both the induction of heat shock proteins (98, 85, 70 kD) and the intracellular pH, determined by means of 31P-NMR spectroscopy, show a similar dose response to increasing temperature or concentrations of 2,4-dinitrophenol (DNP). Temperature increases from 23 degrees to 32 degrees C or more, or concentrations of DNP higher than 1 mM cause a significant increase in the synthesis rate of heat shock proteins and a significant decrease of the intracellular pH. A similar correlation is found in a mitochondrial mutant (Q) defective in oxidative phosphorylation. Intracellular signal transduction may thus involve H+-concentration changes independent of intact oxidative phosphorylation.

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 reviewed. 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. (Chronobiology International, 18(3), 329–369, 2001)

Circadian rhythms and protein turnover: The effect of temperature on the period lengths of clock mutants simulated by the Goodwin oscillator

Recent reports that the circadian clock in both Drosophila and Neurospora consists of a negative feedback loop between clock gene (per, frq) activity and amount of clock protein make the Goodwin oscillator a timely model for circadian rhythms. This model is characterized by a negative feedback loop in the clock gene expression with synthesis/degradation reactions associated with each of the intermediates in the loop. The model predicts that predominantly degradation processes of clock mRNA or clock protein control the circadian period and temperature compensation. Assuming a turnover homeostasis of the clock protein, the model explains temperature effects on period length in per and frq mutants. Recent results obtained in Drosophila [1] and Neurospora [2] show that the circadian clock consists basically of a negative feedback loop. This makes the Goodwin model [3, 4] (Fig. 1) and related oscillators [5] useful models for kinetic studies of circadian rhythms. The Goodwin oscillator shows many properties also observed in circadian clocks, such as temperature compensation [6, 7], phase response curves for temperature steps and pulses, and entrainment by tern-

Heat shock protein induction by certain chemical stressors is correlated with their cytotoxicity, lipophilicity and protein-denaturing capacity

Seven agents were analyzed with respect to their ability to induce heat shock protein (HSP) synthesis in C6 rat glioma cells. Induction of HSP synthesis was correlated with cytotoxicity and lipophilicity of the substances. In addition to the first four n-alcohols (methanol, ethanol, propanol and butanol) and phenol, whose capacity to induce HSP was analyzed earlier (Neuhaus-Steinmetz et al., 1994. Mol. Pharmacol. 45, 36-41), isopropanol, 1,4-dinitrophenol (DNP), diethylstilbestrol (DES), carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, paracetamol and acetyl salicylic acid (ASA) induced HSP synthesis after a 1-h incubation at a substance-specific concentration. The maximal induction of HSPs was closely correlated with the cytotoxicity of all substances and occurred when cell viability was reduced to 75 +/- 11% of the controls. Cytotoxicity and the ability to induce HSP were correlated with the lipophilicity of the alcohols, phenol, rotenone and paracetamol. Calculation of the hypothetical membrane concentrations of these compounds yielded a nearly equal value (0.54 +/- 0.13 M), indicating that interaction of substances with lipophilic cellular compounds, such as membranes or lipophilic core regions of proteins, is a critical step leading to HSP induction. This assumption is supported by a correlation between HSP induction and protein denaturation by the different alcohols (Herskovits et al., 1970. J. Biol. Chem. 245, 2588-2598). We assume that the amount of misfolded proteins induced by these lipophilic agents is responsible for the induction of HSP synthesis. ASA, DNP and CCCP induced HSP at lower concentrations than substances with a similar lipophilicity, which may be due to effects which add to the misfolding of proteins or to other signal pathways.

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.

Phase response curves obtained by perturbing different variables of a 24 hr model oscillator based on translational control

Abstract We have used computer analysis of a modified version of a model oscillator ( Goodwin, 1965 ) to simulate phase response curves such as are obtained in circadian systems. In accordance with recent working hypotheses of the mechanism of the circadian clock, the model consists of three components: an enzyme, its product and a translational inhibitor whose activity depends on the translational product, which in turn determines the rate of enzyme synthesis. The oscillations of the three variables were perturbed with single impulses applied at different phases. The phase and amplitude of the resulting curves depended on the variable affected, the sign of the perturbing stimulus and its strength. The consequences of these results for interpreting the molecular mechanism of circadian clocks on the basis of phase response curves to various treatments are discussed.