No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Cellular and Molecular Aspects of Circadian Oscillators: Models and Mechanisms for Biological Timekeeping
Abstract
Several approaches to elucidate the nature of biological clocks, particularly circadian oscillators, have emerged over the years (Hastings and Schweiger 1976; Edmunds 1988). These include the attempt to locate the anatomical loci responsible for generating these periodicities, efforts to trace the entrainment pathway for light signals (and other zeitgebers) from the photoreceptor(s) to the clock itself, the experimental dissection of the clock using chemicals and metabolic inhibitors and employing the exciting new techniques of molecular genetics, and the characterization of the coupling pathways and the transducing mechanisms between the clock(s) and the overt rhythmicities (hands) it drives. The results obtained by these experimental lines of attack, in turn, have provided the grist for several classes of biochemical and molecular model for autonomous circadian oscillators (COs).
... From an evolutionary perspective, the adaptation of an organism's behavior to its environment has depended on one of life's fundamental traits: biological rhythm generation. In virtually all light-sensitive organisms from cyanobacteria to humans, biological clocks adapt cyclic physiology to geophysical time with time-keeping properties in the circadian (24 h), ultradian (<24 h) and infradian (>24 h) domains (Edmunds, 1988; Lloyd, 1998; Lloyd et al., 2001; Lloyd and Murray, 2006; Lloyd, 2007; Pittendrigh, 1993; Sweeney and Hastings, 1960) By definition, all rhythms exhibit regular periodicities since they constitute a mechanism of timing. Timing exerted by oscillatory mechanisms are found throughout the biological world and their periods span a wide range from milliseconds, as in the action potential of neurons and the myocytes, to the slow evolutionary changes that require thousands of generations. ...
... Circadian rhythms, the most intensively studied, are devoted to measuring daily 24 h cycles. A variety of physiological processes in a wide range of eukaryotic organisms display circadian rhythmicity which is characterized by the following major properties (Anderson et al., 1985; Edmunds, 1988): (i) stable, autonomous (self-sustaining) oscillations having a free-running period under constant environmental conditions of ca. 20–28 h; (ii) phase lability, reflected by the ability of the oscillation to reset its phase in response to an environmental stimulus (Zeitgeber); and (iii) conservation of the period (though not the amplitude) of the rhythm at different ambient temperatures within the physiological range. ...
... Conservation of the period of a rhythm at different ambient temperatures within the physiological range is termed temperature compensation, and is one of the most conspicuous features that qualify a rhythm as a clock. The ability to compensate for fluctuations in temperature is precisely what one would anticipate of any clock mechanism in order to be a reliable timekeeper (Edmunds, 1988; Lloyd, 1998; Murray et al., 2001; Pittendrigh, 1993). Elsewhere (Aon et al., 2006) we have shown that under " physiological " conditions mitochondrial ∆Ψ m fluctuates in a non-random (correlated) manner at high frequency within a restricted amplitude range, implying depolarizations of only microvolts to a few millivolts. ...
A mitochondrial oscillator dependent on reactive oxygen species (ROS) was first described in heart cells. Available evidence now indicates that mitochondrial energetic variables oscillate autonomously as part of a network of coupled oscillators under both physiological and pathological conditions. Moreover, emerging experimental and theoretical evidence indicates that mitochondrial network oscillations exhibit a wide range of frequencies, from milliseconds to hours, instead of a dominant frequency. With metabolic stress, the frequency spectrum narrows and a dominant oscillatory frequency appears, indicating the transition from physiological to pathophysiological behavior.
Here we show that in the pathophysiological regime the mitochondrial oscillator of heart cells is temperature compensated within the range of 25–37°C with a Q10 = 1.13. At temperatures higher than 37°C, the oscillations stop after a few cycles, whereas at temperatures lower than 25°C the oscillations are asynchronous. Using our mitochondrial oscillator model we show that this temperature compensation can be explained by kinetic compensation. Furthermore, we show that in the physiological domain temperature compensation acts to preserve the broad range of frequencies exhibited by the network of coupled mitochondrial oscillators.
The results obtained indicate that the mitochondrial network behaves with the characteristics of a biological clock, giving rise to the intriguing hypothesis that it may function as an intracellular timekeeper across multiple time scales.
... Thus, it is not surprising that the dissociation procedure used in this study allowed the recovery of mainly photoreceptors and glial cells and, to a much lesser degree, a few fibroblasts and neurons (red blood cells, very numerous at the end of the dissociation, and macrophages are usually washed out after 2 days of culture). After dissociation, the only cell type clearly identified under the light microscope was the photoreceptor cell, thanks to its typical segmented organization, identical to that of trout pineals after dissociation (see Fig. 2 in %gay et al. , 1992, andFig. 1 in Falcdn et al., 1992b). ...
... This suggests that the different cellular oscillators of the pike pineal have a very similar free running period and that they are strongly coupled with each other. It is generally admitted that coupling among a population of cellular oscillators is of crucial importance for the overall expression of a circadian rhythm (Edmunds, 1988(Edmunds, , 1992. In the pineal organ of the pike, cell junctions (excepting desmosomes) are exclusively seen between photoreceptors (Falch, 1979b). ...
In the pike pineal, the rhythm of melatonin (MEL) secretion is driven by a population of cellular circadian oscillators, synchronized by the 24 h light/dark (LD) cycle. Because the pineal photoreceptor contains both the input and output pathways of the clock, this cell is likely to be a cellular circadian system by itself. To support this idea, we have dissociated and cultured pike pineal cells as well as purified photoreceptors. In culture, the pineal cells reassociated in follicles, surrounded by collagen fibres. At the electron microscopic level, they appeared well preserved. Total cells consisted mainly of photoreceptors and glia. Purified cells corresponded exclusively to photoreceptors. Under LD, MEL production was rhythmic. Under constant darkness (DD), the rhythm was well sustained for at least six 24 h cycles (tau = 24/27 h) with 1 x 10(6) total cells/well or below; with 2 x 10(6) total cells/well, a strong damping occurred towards high levels as soon as after the second cycle. At the density of 0.5 x 10(6) cells/well, purified photoreceptors produced less MEL than an equivalent amount of total cells. However, the pattern of the oscillations was similar to that observed with 2 x 10(6) total cells, i.e. a damping occurred rapidly. Decreasing the density to 0.125 x 10(6) photoreceptors/well resulted in a loss of homogeneity among replicates. The production of melatonin by single photoreceptors was monitored by means of the reverse haemolytic plaque assay. Both under LD and under DD, the number of photoreceptors releasing melatonin was higher during the (subjective) dark than during the (subjective) light. The results provide strong support to the idea that the pike pineal photoreceptor is a cellular circadian system. Expression of the oscillations seemed to depend on several factors, including cell to cell contacts between photoreceptors. There is indication that also MEL and glia might be involved.
... Steenland A (see figure 1): Most of the functions in the body has a rhythm during the day, so that for instance concentration of hormones and lipids, temperature, hand grip strength, airway narrowing etc. each day follow their own pattern (figure 2). These biological and psychological circadian [4] rhythms are driven by an autoregulatory genetic clock 124 in the suprachiasmatic nuclei of the hypothalamus 125 and exists even in dark caves where no zeitgebers [5] as the dark/light pattern are present. The robust clock which in most individuals runs at approximately 25 hour is synchronized to the 24-hour external environment by entrainment of dark/light pattern, watches, social activities etc. Melatonin released from the pineal gland may act as the internal messenger 126 . ...
... These oscillations, known as circadian rhythms, are endogenous because they can occur in constant environmental conditions, e.g. constant darkness [1, 2]. During the last decade experimental advances have shed much light on the molecular mechanism of circadian rhythms [3]. ...
Circadian rhythms which occur with a period close to 24 h in nearly all living organisms originate from the negative autoregulation of gene expression.Deterministic models based on genetic regulatory processes account for theoccurrence of circadian rhythms in constant environmental conditions (e.g.constant darkness), for entrainment of these rhythms by light-dark cycles, and for their phase-shifting by light pulses. At low numbers of protein and mRNA molecules, it becomes necessary to resort to stochastic simulations to assess the influence of molecular noise on circadian oscillations. We address the effect of molecular noise by considering two stochastic versions of a core model for circadian rhythms. The deterministic version of this core modelwas previously proposed for circadian oscillations of the PER protein in Drosophila and of the FRQ protein in Neurospora. In the first, non-developed version of the stochastic model, we introduce molecular noise without decomposing the deterministic mechanism into detailed reaction steps while in the second, developed version we carry out such a detailed decomposition. Numerical simulations of the two stochastic versions of the model are performed by means of the Gillespie method. We compare the predictions of the deterministic approach with those of the two stochastic models, with respect both to sustained oscillations of the limit cycle type and to the influence of the proximity of a bifurcation point beyond which the system evolves to a stable steady state. The results indicate that robust circadian oscillations can occur even when the numbers of mRNA and nuclear protein involved in the oscillatory mechanism are reduced to a few tens orhundreds, respectively. The non-developed and developed versions of the stochastic model yield largely similar results and provide good agreement with the predictions of the deterministic model for circadian rhythms.
... Circadian rhythms impact athletic performance (Smith, et al., 1997) and work performance especially in relation to shift-work schedules (Scott, 1990). At the other end of the spectrum researchers are discovering the genetic, molecular and neural mechanisms by which the biological clock(s) is designed and functions (Ashoff, 1984;Czeisler et al., 1999;Darlington et al., 1998;Dunlap, 1993Dunlap, , 1999Edmunds, 1992;Moore. 1999;Redfern and Lemmer 1997). ...
Biological rhythmicity is a fundamental characteristic of all life forms, from primitive bacteria to man. The molecular biology, genetics, and the neurobiology of the biological clock(s) are being elucidated. Daily (circadian) statistically significant fluctuations occur in all of the normal biological variables studied in the experimental animal and the human. Many researchers, however, are not aware of the negative impact biological rhythmicity can have on experimental design and/or data interpretation. This article serves not as a review, but as a “field guide” to the pitfalls that can occur when research is performed in the absence of an understanding of biological rhythmicity. The major topics discussed are: 1) data transfer from the diurnally in-active/resting/sleeping lab animal to the diurnally active human, 2) frequency of sampling, 3) free-running vs. synchronization, 4) alternating periods of resistance and susceptibility, 5) phase shifting of a rhythm, 6) the assumption that one mean ± S.E. from control animals can be “stretched” across an experimental time span, and 7) plotting data on an “hours after treatment” format vs. a “time of day” format. The hope is that by avoiding the pitfalls, biological time will become an ally in the endeavor to understand human biology. Anat Rec (New Anat) 261:141–152, 2000. © 2000 Wiley-Liss, Inc.
... The word 'circadian' implies that under constant external conditions the rhythms free-run with a period of approximately, but not precisely, twenty-four hours. Recent evidence indicates that the length of the period is controlled by an endogenous circadian oscillator (clock, zeitgeber, pacemaker) (Aschoff, 1981; Edmunds, 1994; Ikonomov et al., 1998). ...
Kidneys are the main organs regulating water-electrolyte homeostasis in the body. They are responsible for maintaining the total volume of water and its distribution in particular water spaces, for electrolyte composition of systemic fluids and also for maintaining acid-base balance. These functions are performed by the plasma filtration process in renal glomeruli and the processes of active absorption and secretion in renal tubules, all adjusted to an 'activity-rest' rhythm. These diurnal changes are influenced by a 24-hour cycle of activity of hormones engaged in the regulation of renal activity. Studies on spontaneous rhythms of renal activity have been carried out mainly on humans and laboratory animals, but few studies have been carried out on livestock animals. Moreover, those results cover only some aspects of renal physiology. This review gives an overview of current knowledge concerning renal function and diurnal variations of some renal activity parameters in livestock, providing greater understanding of general chronobiological processes in mammals. Detailed knowledge of these rhythms is useful for clinical, practical and pharmacological purposes, as well as studies on their physical performance.
... Several approaches to the elucidation of the nature of biological clocks, particularly circadian oscillators, have emerged over the years (Edmunds 1994). Circadian clocks are time-keeping systems that allow _________________________________________ Giuseppe Piccione, Dipartimento di Scienze Sperimentali e Biotecnologie Applicate, Laboratorio di Cronofisiologia Veterinaria, Facoltà di Medicina Veterinaria, Università degli Studi di Messina, Polo Universitario dell'Annunziata, 98168 Messina, Italy giuseppe.piccione@unime.it ...
To further understanding of the multiple temporal relationships of the physiological process, we monitoredsimultaneously 25 different variables in individual cows. We used 6 Bruna Italiana non – pregnant and non –lactating cows from the same farm. The animals were housed individually in a 12 m2 box under natural 14/10light/dark cycle. They were fed twice daily and water was available ad libitum. Locomotor activity and heartrate were recorded continuously. The rectal temperature, respiratory rate and blood samples were recordedevery 4 hours for 24 consecutive hours. To describe the periodic phenomenon analytically we applied atrigonometric statistical model according to the single cosinor procedure. Twelve of the 25 variables studiedshowed a daily rhythm: locomotor activity, rectal temperature, respiratory rate, haemoglobin, glucose,creatinine, urea, total cholesterol, total lipids, non-esterified fatty acid (NEFA), phosphorus and magnesium.Our results contribute to the understanding of the capacity for reaction and adaptation of animals to theenvironment, and to the improvement in their output by intervention in their environmental circumstances andin the breeding process.
... Of particular interest is the interaction between cellular clocks -both circadian and ultradian -and the eukaryotic cell cycle. There are many examples of temporal control exerted by the circadian clock (for review see Edmunds, 1988), which 'gates' cell division. This means that cell division is confined to a certain phase of the day; a cell that has not attained other requirements, e.g. a minimum cell size, towards the end of the gate must wait until the next day to divide. ...
An ultradian clock operates in fast growing cells of the large ciliate, Paramecium tetraurelia. The period of around 70 minutes is well temperature-compensated over the temperature range tested, i.e. between 18 degrees C and 33 degrees C. The Q10 between 18 degrees C and 27 degrees C is 1.08; above 27 degrees C there is a slight overcompensation. The investigation of individual cells has revealed that two different cellular functions are under temporal control by this ultradian clock. First, locomotor behaviour, which is an alternation between a phase of fast swimming with only infrequent turning, and a phase of slow swimming with frequent spontaneous changes of direction. In addition, the ultradian clock is involved in the timing of cell division. Generation times are not randomly distributed, but occur in well separated clusters. At all of the six temperatures tested, the clusters are separated by around 70 minutes which corresponds well to the period of the locomotor behaviour rhythm at the respective temperatures. Whereas the interdivision times were gradually lengthened both above and below the optimum growth temperature, the underlying periodicity remained unaffected. Also cells of different clonal age had identical periods, suggesting that neither the differences in DNA content, not other changes associated with ageing in Paramecium have an effect on the clock. A constant phase relationship was observed between the rhythm in locomotor behaviour and the time window for cell division; this strongly suggests that the same ultradian clock exerts temporal control over both processes.
... IntroductionEdmunds,Jr, 1988). The effect of light has been studied extensively in many circadian systems, for example by determining the pacemaker response to light pulses delivered at different times in the circadian cycle (the phase response curve; Johnson, 1990). ...
Light is the dominant environmental cue that provides temporal information to circadian pacemakers. In Drosophila melanogaster some period gene mutants have altered free-running circadian periods but entrain to 24 h light-dark cycles. To address the mechanism of light entrainment in Drosophila, we examined the effects of constant light on the period gene (per) and timeless gene (tim) products in wild-type and perS flies. The results indicate that light affects three features of the PER-TIM program: PER and TIM phosphorylation, PER and TIM accumulation, and per and tim RNA cycling. A post-transcriptional effect on the PER-TIM complex is the likely primary clock target, which then delays the subsequent decrease in per and tim RNA levels. This is consistent with a negative feedback loop, in which the PER-TIM complex contributes to the decrease in per and tim RNA levels, presumably at the transcriptional level. There are enhanced constant light effects on the perS mutant, which further support negative feedback as well as support its importance to entrainment of these flies to a 24 h cycle, far from their intrinsic period of 19 h. The perS mutant leads to a truncated protein accumulation phase and a subsequent premature perS RNA increase. A standard 24 h light-dark cycle delays the negative feedback circuit and extends the RNA and protein profiles, compensating for the accelerated RNA increase and restoring the rhythms to wild-type-like periodicity.
A review is given on the properties of glycoconjugates subjected to circadian rhythms: proteoglycans, glycosylated hormones, enzymes, glycolipids. Membrane receptors and their ligands which appear to be frequently glycosylated can play a role in circadian rhythmicity. Circadian modulation of the glycoconjugates metabolism and the effect of glycosylation inhibitors on the circadian rhythms will be discussed. Rhodopsin, a photoreceptor, is glycosylated and presents an unusual type of glycosylation. The role of ceramides as second messengers has been demonstrated and could interact with metabolites important for the rhythm. The formation of a spatial structure of macromolecules such as helicoids is a circadian or ultradian process. The importance of circadian rhythms in cancer has been reported in a few cases and suggests a critical analysis of many reports.
It has become clear over the past years that the circadian system in all organisms is based on molecular processes within single cells, and an unprecedented surge of research has revealed the molecular details of a transcriptional/translational feedback loop that is considered as the molecular clock works. In spite of this excellent progress, central questions are still unanswered: what makes the loop turn over with the long circadian period? How is the loop compensated for extra-and intracellular noise and different levels of metabolism (e.g., temperature compensation)? How do different clocks communicate and couple and how does the system adapt to changing conditions in development, in the environment, in life history or in seasons. Although the complexity of the feedback loop is increasing with every new insight, it still represents a fairly simple molecular network. Many decades of physiological and biochemical research in unicellular organisms shows, however, that the circadian system is highly complex on one hand and exquisitely compensated on the other—qualities that are as yet not entirely explained by the molecular loop. The results gained in unicellular and simple organisms can be used to understand the functioning and the constraints of single cell clocks within complex organisms. A comparison between single cell clocks, be it unicells or in cells of complex organisms can be used to outline general circadian principles that apply to both.
The relationship between the degree of chilling resistance and phase shifting caused by low-temperature pulses was examined in two circadian rhythms in cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12∶12 h at 33° C. The seedlings showed a circadian rhythm of chilling resistance and of cotyledon movement. A pulse of 19° C for 12 h during the chilling-sensitive phase (light period) caused a phase delay of 6 h, while a similar temperature pulse during the chilling-resistant phase (dark period) did not cause any phase shift. Exposure to 19° C, 85% RH (relative humidity) for 12 h during the dark period induced chilling resistance in the following otherwise chilling-sensitive light period. In this light period a 12-h 19° C pulse did not cause a phase shift of chilling resistance. Pulses of low temperatures (5-19° C) were more effective in causing phase delays in the rhythm of cotyledon movement when given during the chilling-sensitive phase than when given during the chilling-resistant phase. A 12-h pulse of 5° C, 100% RH during the light period caused a phase delay of cotyledon movement of 12 h. However, when that pulse had been preceded by a chill-acclimating exposure to 19° C, 85% RH for 12 h during the dark period the phase delay was shortened to 6 h. The correlation between higher degree of chilling resistance and the prevention or shortening of the phase delay caused by low temperatures indicates that the mechanism that increases chilling resistance directly or indirectly confers greater ability for prevention of phase shifting by low temperatures in circadian rhythms.
The equatorial Pacific has been intensively studied since 1989 when Joint Global Ocean Flux Study (JGOFS) promoted an international study of this region [JGOFS, 1990]. Attention was focused on the equatorial Pacific for several reasons. Upwelling in this region brings nutrients into the euphotic zone over a wide area. It also brings up waters with high inorganic carbon content which cause CO2 outgasing to the atmosphere.
Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms.
The purpose of this chronoepidemiologic study was to investigate the time-relationships between the yearly variations in occurrence of violent suicide in Belgium and the yearly variations in various biochemical, metabolic and immune variables in the Belgian population. The weekly mean number of deaths due to violent suicide for all of Belgium for the period 1979-1987 was computed. Twenty-six normal volunteers had monthly blood samplings during one calendar year for assays of plasma L-tryptophan (L-TRP), competing amino acids (CAA), and melatonin levels, maximal [3H]paroxetine binding to platelets, serum total cholesterol, calcium, magnesium, and soluble interleukin-2 receptor concentrations, and number of CD4+ T, CD8+ T and CD20+ B lymphocytes. The annual rhythm in violent suicide rate is highly significantly synchronized with the annual rhythms in L-TRP, [3H]paroxetine binding, cholesterol, calcium, magnesium, CD20+ B cells, and CD4+/CD8+ ratio; the mean peak (violent suicide, [3H]paroxetine binding) or nadir (all other variables) occurs around 3 May. There were significant inverse time-relationships between the time series of violent suicide rate and L-TRP, L-TRP/CAA ratio, total cholesterol, calcium and magnesium, CD4+/CD8+ T cell ratio and number of CD20+ B cells. Maximal [3H]paroxetine binding to platelets was significantly and positively related to the time series of violent suicide. An important part (56.4%) of the variance in mean weekly number of violent suicide rate was explained by the time series of L-TRP, cholesterol and melatonin.
The properties of the melatonin-generating system of a tropical teleost, the sailfin molly (Poecilia velifera), were investigated in vitro in a series of experiments using static or perifusion culture techniques. The properties examined included photic entrainment, circadian rhythmicity under continuous light (LL) and continuous darkness (DD), functionality of the melatonin-generating system at birth, and presence of multiple circadian oscillators in the molly pineal. Pineal glands or skull caps with the pineal gland firmly attached were dissected from adult and new-born fishes, respectively, and placed into static or perifusion culture at constant temperature (27 degrees C) depending upon the experiment. Melatonin release in samples was quantified by RIA. Rhythmic melatonin release was observed from isolated adult pineals under 12L:12D and 14L:10D, with low amounts of melatonin released during the light and high amounts during the dark. Melatonin release was inhibited by LL. However, under DD, melatonin release was robust and rhythmic with a circadian period (Tau) that ranged between 21.3 and 27.0 h (n = 21). Pineals from new-born (1-day old) mollies released melatonin rhythmically under a light:dark cycle and DD in both static and perifusion culture. Melatonin release from half and quarter pineals of adult mollies under DD was robust and rhythmic with circadian periods that ranged between 22.5 and 29.0 h (n = 19). Taken together, these data show that the molly pineal is photosensitive, fully functional from birth, and contains multiple circadian oscillators (at least four) regulating melatonin production.
Blind subterranean mole rats retain a degenerated, subcutaneous, visually blind but functionally circadian eye involved in photoperiodic perception. Here we describe the cloning, sequence, and expression of the circadian Clock and MOP3 cDNAs of the Spalax ehrenbergi superspecies in Israel. Both genes are relatively conserved, although characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of humans and mice, and is different in amino acid composition from that of rats. We also show that MOP3 is a bona fide partner of Spalax Clock and that the Spalax Clock/MOP3 dimer is less potent than its human counterpart in driving transcription. We suggest that this reduction in transcriptional activity may be attributed to the Spalax Clock glutamine-rich domain, which is unique in its amino acid composition compared with other studied mammalian species. Understanding Clock/MOP3 function could highlight circadian mechanisms in blind mammals and their unique pattern as a result of adapting to life underground.
Ideas of homeostasis derive from the concept of the organism as an open system. These ideas can be traced back to Heraclitus. Hopkins, Bernard, Hill, Cannon, Weiner and von Bertalanffy developed further the mechanistic basis of turnover of biological components, and Schoenheimer and Rittenberg were pioneers of experimental approaches to the problems of measuring pool sizes and dynamic fluxes. From the second half of the twentieth century, a biophysical theory mainly founded on self-organisation and Dynamic Systems Theory allowed us to approach the quantitative and qualitative analysis of the organised complexity that characterises living systems. This combination of theoretical framework and more refined experimental techniques revealed that feedback control of steady states is a mode of operation that, although providing stability, is only one of many modes and may be the exception rather than the rule. The concept of homeodynamics that we introduce here offers a radically new and all-embracing concept that departs from the classical homeostatic idea that emphasises the stability of the internal milieu toward perturbation. Indeed, biological systems are homeodynamic because of their ability to dynamically self-organise at bifurcation points of their behaviour where they lose stability. Consequently, they exhibit diverse behaviour; in addition to monotonic stationary states, living systems display complex behaviour with all its emergent characteristics, i.e., bistable switches, thresholds, waves, gradients, mutual entrainment, and periodic as well as chaotic behaviour, as evidenced in cellular phenomena such as dynamic (supra)molecular organisation and flux coordination. These processes may proceed on different spatial scales, as well as across time scales, from the very rapid processes within and between molecules in membranes to the slow time scales of evolutionary change. It is dynamic organisation under homeodynamic conditions that make possible the organised complexity of life.
Definition of the temporal characteristics of cardiovascular rhythms is of scientific interest. This article reviews the literature on the rhythmic pattern of some cardiovascular parameters in domestic animals, providing greater understanding of general chronobiological processes in mammals. The techniques of the chronophysiological studies have been applied in domestic species to determine the existence of periodicity in the cardiovascular functions. Detailed knowledge of these rhythms is useful for clinical, practical and pharmacological purposes and physical performances.
Steady-state mRNA levels of nuclear (rbcS, cab, tubA) and plastid (rbcL, psbA) encoded genes were determined in tomato leaves of different developmental stages. Transcripts were analyzed at four-hour intervals throughout a diurnal cycle in 4 cm-long terminal leaflets, while mRNA levels of the chlorophyll a/b-binding protein (cab), and the small and large subunit of RuBPC/Oase (rbcS, rbcL) are high. At different time points during the day the mRNAs accumulate to characteristic levels. Minor fluctuations of such mRNA levels were determined in the case of rbcS, rbcL, psbA and tubA, while significant alterations are observed for the chlorophyll a/b-binding protein transcript levels. LHCP II transcripts accumulate during the day, reach highest levels at noon and decline to non-detectable levels at 5 a.m. The cab mRNA fluctuates with a periodic length of approximately 24 hours suggesting the existence of a circadian rhythm ("biological clock"), which is involved in gene activation and inactivation. The mRNA oscillation with the same periodic length, but altered amplitude, continues to be present in plants which are kept under extended dark or light conditions. Different mRNA fluctuation patterns are observed for rbcS, rbcL, and psbA under such experimental conditions.
Oscillations in adenosine 3′,5′-cyclic monophosphate (cyclic AMP) level have been proposed to be part of the biochemical feed-back loop(s), or ‘clock(8)’, believed to underlie circadian rhythmicity. This possibility has been examined for a cellular circadian oscillator in synchronously dividing (or nondividing) cultures of the photosynthesis- deficient ZC mutant of the alga Euglena gracilis Klebs (Z). We have demonstrated a bimodal, autonomously oscillating, circadian variation of cyclic AMP content in this unicell. Rhythmic changes of the cyclic AMP level, which may reflect the transition of the cell population through the different phases of the cell division cycle (CDC) in division-phased cultures, also persisted after the culture medium had become limiting and the cells had stopped dividing. We have also shown that the free-running, circadian oscillation of cyclic AMP content displayed by nondividing cells in continuous darkness could be phase-shifted by a light signal (a property inherent to most circadian systems), in a manner that could be predicted from the phase-response curve previously obtained for the cell division rhythm in the ZC mutant. These results suggest a possible role for cyclic AMP, either as an element of the coupling pathway for the control of the CDC by the circadian oscillator, or as a ‘gear’ of the clock itself.
The pacemaker role of the suprachiasmatic nucleus in a mammalian circadian system was tested by neural transplantation by using a mutant strain of hamster that shows a short circadian period. Small neural grafts from the suprachiasmatic region restored circadian rhythms to arrhythmic animals whose own nucleus had been ablated. The restored rhythms always exhibited the period of the donor genotype regardless of the direction of the transplant or genotype of the host. The basic period of the overt circadian rhythm therefore is determined by cells of the suprachiasmatic region.
The period (per) locus, which controls biological rhythms in Drosophila, was originally defined by three chemically induced mutations. Flies carrying the pero mutation were arrhythmic, whereas pers and perl mutants had circadian behavioural rhythms with 19-hour and 29-hour periodicities, respectively. Wild-type flies have 24-hour rhythms. Here we compare the per locus DNA sequences of the three mutants with the parental wild-type. The pers and perl mutations lead to amino-acid substitutions, whereas pero introduces an early translation stop (amber). The results indicate that the protein product of per controls biological rhythms. We also report that the abundance of this protein may set the pace of the Drosophila clock. Although circadian rhythms are restored when arrhythmic (per-) Drosophila are transformed with per locus DNA, flies receiving identical transforming DNA segments can produce rhythms with periods that differ by more than 12 hours. Transcription studies reveal a tenfold variation in the level of per RNA among transformed lines. Levels of per RNA are inversely correlated with period length, so that flies with lowest levels of the per product have slow-running biological clocks. On the basis of the combined studies we suggest that perl and pers mutants produce hypoactive and hyperactive per proteins, respectively.
The fruit fly Drosophila melanogaster displays an ovarian diapause that is regulated by photoperiod. Newly eclosed female flies (Canton-S wild type) exposed to short days (less than 14 hr of light per day) at 12 degrees C (or 10 degrees C) enter a fairly shallow reproductive diapause. Females exposed to long days (16 hr of light per day) at the same low temperature undergo ovarian maturation. The short day induced diapause continues for 6-7 weeks under a 10:14 light/dark cycle at 12 degrees C but is terminated rapidly after a transfer to higher temperature (18 or 25 degrees C) or to long days (18:6 light/dark cycle). Females from three strains homozygous for alleles of the period (per) locus, reportedly arrhythmic for behavioral circadian rhythms, and females that possessed two overlapping deletions of per were also capable of discriminating between long and short days, although, when compared with the wild-type flies, the critical day length was shifted to shorter values by approximately 2 hr. It is concluded that the period locus is not causally involved in photoperiod time measurement.
The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels. 1988 Deutsche Botanische Gesellschaft/German Botanical Society
The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker that regulates many circadian rhythms in mammals. Experimental work in microorganisms and invertebrates suggests that protein synthesis is required for the function of the circadian oscillator, and recent experiments in golden hamsters suggest an acute inhibition of protein synthesis can induce phase shifts in a mammalian circadian pacemaker. To determine whether protein synthesis in the SCN region is involved in the generation of circadian rhythms in mammals, a protein synthesis inhibitor, anisomycin, was microinjected into the SCN region, and the effect on the circadian rhythm of locomotor activity of hamsters was measured. A single injection of anisomycin into the SCN region induced phase shifts in the circadian activity rhythm that varied systematically as a function of the phase of injection within the circadian cycle. These results suggest that protein synthesis may be involved in the generation of circadian rhythms in mammals and that the anatomic site of action of anisomycin is within the hypothalamic suprachiasmatic region.
The isolated Bulla eye expresses a circadian rhythm in optic nerve impulse frequency. In an effort to determine the anatomical location of the circadian pacemaking system within the retina we surgically reduced the eye. We report that: 1. The approximately 1000 large photoreceptors which form a cell layer immediately surrounding the lens, are not required for the expression of a circadian rhythm. Eyes which are surgically reduced so that only the basal retinal neuron population remains, continue to express a circadian rhythm indistinguishable in period to intact eyes. 2. The photoreceptor layer is also not required for light-induced phase shifts of the ocular rhythm. Retinal fragments containing only basal retinal neurons can be phase advanced or delayed by 6 h light pulses provided at the appropriate circadian phase. 3. Of the approximately 100 basal retinal neurons in the Bulla eye, only a small proportion are required for the expression of a circadian rhythm in optic nerve frequency. Ocular fragments with as few as 6 basal retinal neuron somata remain rhythmic, and exhibit a free-running period indistinguishable from intact eyes. 4. Intact basal retinal somata are required for the expression of a circadian rhythm in optic nerve impulse frequency. Retinal fragments consisting of an optic nerve with a small amount of neuropil region produce spontaneous action potentials without evidence for a circadian modulation. 5. An explicit model for the organization of the circadian pacemaker system in the Bulla retina is proposed.
Living in our hectic, jet-set and jet-lag society, most of us have experienced the discomforts imposed by a rapid transfer into shifted day-night cycles, differing in phase considerably from the ones we are used to. Our bodies usually need some days to readjust to the new environment, before our endogenous rhythms have accommodated the changed conditions.
Progress in a particular field of biology has often been the result of the development of an organism or system especially well suited to research on that problem. The value of E. coli to molecular biology, the mammalian red blood cell to membrane biochemistry, and the oat coleoptile to plant physiology has been well-documented.
We reared wild-type flies in constant darkness for several generations, subculturing and handling them under a red safelight known not to affect Drosophila clock function or to be perceived visually by them. The locomotor activity rhythms of newly eclosed individuals were subsequently monitored in constant darkness, with no further exposure to light other than the far-red safelight. Only 23% of the flies had wild-type activity rhythms, and half of these had strong ultradian components, uncharacteristic of normally reared wild-type flies; an additional 10% had a novel phenotype: weak, short-period (20- to 22-h) circadian rhythms and relatively strong ultradian rhythms The remaining 68% were phenocopies of the period mutants, per°-like, but apparently arrhythmic, lacking significant rhythmicity that could be discovered by our robust criteria; another 16% were per°-like with significant ultradian rhythms. The balance (16%) were phenocopies of , with relatively weak long-period rhythms and multiple ultradian rhythms. We hypothesize that exposure to light at some point in development acts to couple a population of ultradian oscillators into a composite circadian clock, and that the disruption of the rhythms seen in DD-reared animals is functionally identical to that found in animals mutant for the period gene.
Phase shifting by light of the circadian conidiation rhythm of the Neurospora crassa strain band, of the riboflavin-deficient double mutant band rib2 and of the temperature-sensitive double mutant band ribl was measured. Fluence response curves of the band strain exhibited two distinct steps, whereas those of band ribl and band rib2 revealed only one step. Maximum phase advances observed were 5.5 h in band and 10.4 h in the band rib strains. Sensitivity of band rib2 to light was proportional to the riboflavin concentration in the growth medium over a 100 fold range. Extracellular flavin in the medium did not sensitize the strains. Riboflavin applied after exposure to light showed no effect. Light sensitivity correlated with the level of cellular riboflavin. Four analogs of riboflavin, none of which can be phosphorylated, increased the sensitivity of Neurospora to light. Even at high riboflavin concentrations in the medium, the sensitivity of the band rib2 strain to light was not saturated. In addition, four riboflavin derivatives with bulky substituents at positions 3, 8 or 10 of the isoalloxazine nucleus sensitized both strains. From our data, we conclude, that a) a cellular flavin controls the sensitivity of Neurospora crassa to light; b) that this flavin compound is riboflavin; and c) that the active riboflavin is not protein bound.
Leaflet movements in the legume Samanea saman are under joint control by light and a circadian oscillator. The movements are driven by massive fluxes of K⁺, Cl⁻, and H⁺ through pulvinar motor cell membranes. Light and the oscillator affect leaflet movements by altering the activity of ion transport systems. Some effects of light on ion transport may be mediated by the phosphatidylinositol (PI) cycle, since brief irradiation of the pulvinus with white light accelerates PI turnover. 1988 Deutsche Botanische Gesellschaft/German Botanical Society
Characteristic steady-state mRNA level oscillations were monitored for the chlorophyll a/b-binding (cab) protein in tomato plants grown under the natural day/night (light/dark) regime as well as under constant environmental conditions. This typical expression pattern was altered when plants were transferred to different light/dark regimes. For example, by shifting the light phase by six hours, a change of the time points of maximum and minimum of expression level was monitored, while the principal oscillation pattern remained the same. It appeared that the transition from dark to light is involved in determining the time points of minima and maxima of mRNA accumulation.
After exposing tomato plants to an abnormal light/dark periodicity (e.g. six hours of alternating light/dark) an altered oscillation pattern was determined: within 24 hours two maxima of cab mRNA levels were detected. However, this ‘entrained’ abnormal rhythm was not manifested at the molecular level and the circadian pattern reappeared under constant environmental conditions (e.g. darkness). This result favours the hypothesis that the oscillation pattern of the cab mRNA in tomato plants is not only endogenous but also hereditary.
Under continuous illumination this unicellular aerobic cyanobacterium fixes dinitrogen continuously at a variable and usually low rate. Exposure of the culture to diurnal light/dark cycles invariably results in the virtual restriction of nitrogenase activity to the dark periods. The rhythmic diurnal dinitrogen fixation pattern becomes a truely endogenous cycle which persists for at least 4 days with decreasing magnitude on exposing the culture to continuous illumination. The free running time of the rhythm appears to decrease from an initial 26 h to 22 h in the course of 4 days. This appears to be the first record of an endogenous rhythm in a prokaryote.
The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels.
Synchronous cultures of the unicellular alga Chlamydomonas reinhardii established by repetitive light and dark cycles displayed both ultradian and circadian rhythms in chlorophyll a levels. The ultradian oscillation had a period of approximately 55 min and appeared superimposed upon the circadian rhythm. These data support the theory that the ultradian oscillator may be a common element in both circadian and cell cycle timing.
A small amount of translatable mRNA for a nuclear-coded precursor apoprotein of the LHC-I (light-harvesting complex of photosystem I) with a molecular mass of 26 kDa is present in etiolated bean leaves. The expression of this protein is phytochrome-controlled and follows circadian oscillations, for the appearance of which a red-light pulse is sufficient. The rhythmical oscillations persist for many hours in the dark following the red-light pulse or in continuous white light. The similarity of this rhythm in the light-induced accumulation of LHC-I mRNA with that of LHC-II mRNA [(1989) Plant Physiol. 90, 665] suggests that the same oscillator may govern the expression of all chloroplast protein genes regulated by light.
Protein synthesis seems to be a general requirement for circadian timing. Defining the time period when inhibition of protein synthesis changes the phase of the biological clock may help identify proteins that are involved in the molecular mechanism of circadian timing.
Rothman and Strumwasser (1976), Jacklet (1977), and Lotshaw and Jacklet (1986) gen erated phase response curves (PRCs) for relatively long pulses (6 hr) of anisomycin and puromycin administered to Aplysia eyes. Using somewhat different conditions, we generated a 4-hr anisomycin PRC from Aplysia eyes and found that our anisomycin PRC was similar to that previously described by Lotshaw and Jacklet (1986). We studied recovery of protein synthesis after 1-hr and 6-hr anisomycin treatments and found recovery to be very slow; from 8 to 12 hr appeared to be required for full recovery after anisomycin. Slow recovery occurred when eyes were treated either in buffered artificial seawater or in enriched culture media. Because of the slow recovery after anisomycin, it is difficult to infer accurately from the anisomycin PRC when protein synthesis is important.
To identify an inhibitor whose effect reverses quickly, we studied recovery from inhibi tion of protein synthesis after emetine, L-O-methylthreonine, and cycloheximide. Both eme tine and L- O-methylthreonine seemed to reverse no faster than anisomycin, but cycloheximide reversed faster than all the other inhibitors. Cycloheximide (10 mM, 1 hr) produced 89% inhibition of [ ³ H] leucine incorporation, and within 3 hr after removal of cycloheximide, the recovery was 85%.
A PRC was obtained using 1-hr treatments of cycloheximide (10 mM). Cycloheximide did not significantly phase-shift from circadian time (CT) 8 to CT 20, and cycloheximide delayed (by about 1 hr or less) the circadian rhythm from CT 20 to CT 8. The cycloheximide PRC was not due to different kinetics of recovery at different phases, as evidenced by similar recovery times when recovery from inhibition by cycloheximide was measured at two phases (a phase when cycloheximide produced no phase shift and a phase when cycloheximide delayed the rhythm).
P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.
In Neurospora crassa, the cel mutation lengthens the period of the circadian rhythm when the medium is supplemented with linoleic acid (18:2). Double mutant strains were constructed between cel and the clock mutants prd-1 and four alleles at the frq locus. It was found that: (1) the effect of 18:2 on cel was blocked by prd-1, i.e., prd-1 is epistatic to cel. (2) cel and frq interact such that the percent increase in the period produced by 18:2 was inversely proportional to the period of the frq parent. (3) Data from the literature on period effects in double mutant strains support a multiplicative rather than an additive model. A biochemical interpretation of these interactions is discussed, based on the control of flux through metabolic pathways. Because the cel strain is known to be deficient in the pantothenate derivative normally attached to the fatty acid synthetase (FAS) complex, the possibility that cel may affect other pantothenate-modified proteins was investigated. It was found that in the cel/sup +/ strain, five proteins of molecular weights (M/sub r/) 9000, 19,000, 22,000, 140,000, and 200,000 were labelled with (/sup 14/C)pantothenate. In the cel strain, only the 200 k (FAS) label was reduced in amount. Therefore, there is no evidence that cel affects circadian rhythmicity through any deficiency other than FAS. A biochemical model for circadian rhythmicity in Neurospora is presented. Oscillations in cytoplasmic and mitochondrial Ca/sup 2 +/ are proposed; clock mutations are postulated to affect Ca/sup 2 +/ transporters and the mitochondrial membrane; and phase-shifting effects are accounted for by changes in Ca/sup 2 +/ or ATP levels.
Among nitrogen-fixing microorganisms, nitrogen-fixing cyanobacteria are unique in their ability to carry out oxygen-evolving photosynthesis and oxygen-labile nitrogen fixation within the same organisms1–3. These seemingly incompatible reactions take place in heterocystous cyanobacteria by the spatial separation of the site of nitrogen fixation (heterocysts) from the site of photosynthesis (vegetative cells)4,5. Several hypotheses have been proposed to explain these mechanisms in non-heterocystous cyanobacteria3,6–11. Using batch cultures of Gloeothece (Gloeocapsa) spp., Gallon and collaborators demonstrated the mechanism of temporal separation of photosynthesis and nitrogen fixation into the light and dark periods of growth, respectively9. However, the mechanisms by which these two incompatible reactions can occur under continuous light conditions still remained ambiguous. Using novel strains of aerobic nitrogen-fixing, unicellular marine cyanobacteria, Synechococcus spp., grown under synchronized conditions, we report here that nitrogen fixation and photosynthesis occur at different phases in the cell division cycle. Our data, obtained under both diurnal light/dark cycle and continuous illumination, indicate that the temporal separation of the two phases during the cell division cycle is the mechanism by which these unicells can grow photoautotrophically under nitrogen-fixing conditions.
Discusses basic properties of circadian systems, describes clinical implications of disruptions of biological clocks (internal temporal programs), and reviews current experimental work on the biochemical mechanisms of biological clocks. One strategy involves the investigation of pharmacological agents altering the clock, while another approach includes delineating the pathway of entrainment by light. Molecular genetics promises new insight into circadian rhythms, now being studied through the unicellular alga
Gonyaulax. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C2H4 and 1.4 mg O2 mg Chl a
-1h-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C2H4 mg Chl a
-1h-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.
Populations ofGonyaulax polyedra, in two different phases, about 11 h apart, were mixed, and the intensity of their spontaneous bioluminescence glow recorded
for about 2 wk under conditions of constant dim (35±3 μE/m2/s) white light and constant temperature (19.0±0.3°C). The phases and amplitudes of glow signals recorded from mixed cultures
were compared with those obtained from the arithmetic sum of the intensity data from two control vials. Peaks in control cultures
generally remained separate, but there was a spontaneous increase in the period beginning 6–11 d after the onset of constant
conditions. This did not occur in cultures in which the medium was exchanged with fresh medium every 2 d. In the actual mixes
of two cultures there was a merging of the two subpeaks in the signal, which did not occur when the medium was exchanged.
The results indicate that conditioning of the medium by cells may affect the period of the circadian rhythm and that this
might result in a type of communication.
Evidence is steadily mounting that the proto-oncogenes, whose products organize and start the programs that drive normal eukaryotic cells through their chromosome replication/mitosis cycles, are transiently stimulated by sequential signals from a multi-purpose, receptor-operated mechanism (consisting of internal surges of Ca2+ and bursts of protein kinase C activity resulting from phosphatidylinositol 4,5-bisphosphate breakdown and the opening of membrane Ca2+ channels induced by receptor-associated tyrosine-protein kinase activity) and bursts of cyclic AMP-dependent kinase activity. The bypassing or subversion of the receptor-operated Ca2+/phospholipid breakdown/protein kinase C signalling mechanism is probably the basis of the freeing of cell proliferation from external controls that characterizes all neoplastic transformations.
In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker ofBulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that:1.
Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization.
2.
Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180° in circadian time to depolarization.
3.
Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection.
4.
The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts.
5.
The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3×10−7M inhibits light-induced phase shifts without blocking the photic response of the BRNs.
The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.
Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.
Many responses of plants to phytochrome involve alterations in gene expression. Recent studies with transgenic plants as an expression system have led to the identification of cis-acting elements that mediate responses to phytochrome and light.
Glutathione (GSH) levels were measured in platelet-rich plasma (PRP) and concentrates at 4 hour intervals during storage. The values fell steadily during the first several hours after collection at 9:00 a.m., reaching the lowest level at midnight, 14 hours later. Subsequently, the levels rose to a new peak value at 4:00 a.m. GSH levels continued to show cyclic variation over the 48 hours examined in this study. Change in schedules of light and dark under which the platelets were stored had no effect on GSH periodicity. Platelets from night shift workers also displayed a similar periodicity to that of day workers. Erythrocytes failed to demonstrate a similar time related variation in GSH levels during storage.
The circadian clock in the unicellular alga Gonyaulax polyedra is accelerated by a substance in extracts from the cells themselves. The extracts have been fractionated using the circadian rhythm of bioluminescence as bioassay. The active substance, termed gonyauline, has been isolated and characterized as a novel low molecular weight cyclopropanecarboxylic acid (S-methyl-cis-2-(methylthio) cyclopropanecarboxylic acid). Synthetic gonyauline has a similar shortening effect on the period of the circadian clock.
Mutations in the period (per) gene of Drosophila melanogaster affect both circadian and ultradian rhythms. Levels of per gene product undergo circadian oscillation, and it is now shown that there is an underlying oscillation in the level of per RNA. The observations indicate that the cycling of per-encoded protein could result from per RNA cycling, and that there is a feedback loop through which the activity of per-encoded protein causes cycling of its own RNA.
The effect of inhibitors of protein synthesis on the phase shifting action of light was investigated. Anisomycin and cycloheximide appeared to block advance phase shifts produced by light. This result suggested that light might phase shift by changing the synthesis of some proteins. Examining proteins separated by two-dimensional gel electrophoresis, we found that incorporation of amino acids into 11 proteins was changed during a 6-h light pulse. Nine of these 11 proteins were affected by light in a phase-dependent manner. Elevated extracellular potassium and 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP), two treatments that mimic effects of light on the rhythm, also changed amino acid incorporation into a number of proteins. All of the five proteins affected by 8-bromo-cGMP were also affected in the same manner by light. Three proteins were affected similarly by elevated potassium, light, and 8-bromo-cGMP. Exposure of eyes to label at different times after light treatment showed that the effects of light on some proteins were long lasting. In addition, some proteins were not affected during light but were affected only several hours after light. Some of the eye proteins affected by light were also altered by serotonin (5-HT), another phase-shifting agent. The proteins affected by light, elevated potassium, 8-bromo-cGMP, and 5-HT are candidates for components of the circadian system either as an element of the entrainment pathway or the oscillator mechanism.
Serotonin (5-HT) shifts the phase of the circadian oscillator of the eye of Aplysia californica in a phase dependent manner. This indicates that 5-HT acts, either directly or through some intermediaries, on a component of the oscillator. Since our goal is to identify the components of the oscillator, we are following the pathway through which 5-HT has its effect on the rhythm. The effect of 5-HT on the rhythm has been shown to be mediated by an increase in intracellular cyclic adenosine-3',5'-monophosphate (cAMP). The most likely action of cAMP is to activate cAMP-dependent protein kinase. Therefore, we used two-dimensional polyacrylamide gel electrophoresis to investigate changes in 32P labelled phosphoproteins which occur with 5-HT and other treatments. Fourteen proteins showed increased incorporation of 32P when eyes were exposed to treatments of 5-HT from CT 06 to 12. Two proteins showed decreased incorporation. 8-bt-cAMP mimicked all but one of the increases and both decreases in incorporation produced by 5-HT. 8-bt-cAMP increased incorporation into three additional proteins and decreased incorporation into three others that were not affected by 5-HT. Incorporation into one protein was increased by 5-HT but decreased by 8-bt-cAMP. By comparison, light, which has little or no effect on the rhythm at this phase, only affected one protein. The protein increased by light was also increased by 5-HT. Tetradecanoic phorbol acetate (TPA), administered during CT 06-12, also had little effect on the rhythm at this phase. TPA increased incorporation into twenty proteins and decreased incorporation into three.(ABSTRACT TRUNCATED AT 250 WORDS)
Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5-9 h) phase shifts of the circadian conidiation rhythm of Neurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989). Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6-9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180 degrees out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0-12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0. Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with 35S-thio gamma-ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42 degrees C) temperatures. Altogether, the results indicate that Ca2(+)-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism of Neurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.
Oscillations in glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD) activities were recorded in suspensions of intact human red blood cells (RBCs) exposed to various light regimens. The periods of these oscillations, defined as "long ultradian," ranged between 13 and 18 h regardless of light regimen. The patterns of enzymatic activities were the same when assayed at each time point, in full hypotonic hemolysates, and membrane-free hemolysates. However, if hemolysates were prepared by sonication the activity pattern did not exhibit significant oscillations and the activity was higher than that recorded in hypotonic hemolysates. The observed rhythms may reflect a time-dependent attachment and detachment of enzyme molecules from cell membrane, suggesting that at the bound state the enzyme molecules are (temporarily) inactive. Oscillations with similar long ultradian periods were also observed in Ca++ concentration of suspended RBCs and in the binding of Ca++45 to human RBC ghosts. Treatment of the RBCs with A2C or Diamide before the preparation of the ghosts changed or distorted the rhythmic pattern of Ca++45 binding. These results point to the role of the membrane in processing the long ultradian oscillations. The relation between this type of oscillations to circadian rhythm is discussed.
The circadian pacemaker in the retina of the eye of the marine snail Bulla gouldiana was examined using the whole eye in vitro preparation. Phase-response curves were generated to 6-h pulses of a low calcium EGTA solution and to a hyperpolarizing low potassium-low sodium solution. Both treatments yielded similar phase response curves with phages delays in the late subjective night/early subjective day and phase advances in the late subjective day. The similarity of the phase response curves to hyperpolarizing and low calcium solutions and the absence of additivity when both treatments are combined raises the possibility that both treatments affect the underlying pacemaker through a common mechanism. The persistence of phase shifts to low calcium pulses delivered in the presence of depolarizing light suggests that hyperpolarization is not required for low calcium phase shifting. However, it is possible that both treatments act by reducing a transmembrane calcium flux which is postulated to result from the periodic depolarization of the pacemaker cell membrane during the subjective day. Since a transmembrane calcium flux is known to be essential for both light and depolarization-induced phase shifting, we discuss the hypothesis that calcium fluxes play a pivotal role in the entrainment pathway of the circadian pacemaker.
The per locus has a fundamental involvement in the expression of biological rhythms in Drosophila. Mutations at this locus can shorten, lengthen or eliminate a variety of rhythmic activities that range from circadian behaviours, exemplified by eclosion and locomotor activities, to short-period behaviour such as the 55-s rhythm of courtship song. DNA from the per locus has been cloned, and we have used P-element-mediated DNA transformation to establish that a 7.1-kilobase(kb) HindIII fragment contains a functional copy of the gene. This transforming DNA contains a single transcription unit which gives rise to a 4.5-kb poly(A)+ RNA. Here we report the results of a search for sequences homologous to the per locus DNA in the genomic DNA of several species of vertebrates. An unusual, tandemly repeated sequence forming a portion of the 4.5-kb per transcript is homologous to DNA in chicken, mouse and man. Cloned DNAs from the mouse and Drosophila are related by long, uninterrupted tandem repetitions of the sequence ACNGGN. At the per locus, these tandem repeats are predicted to code for poly(Thr-Gly) tracts up to 48 amino acids long. These repeated sequences are also transcribed in the mouse. Several long tracts of poly(Thr-Gly) appear to be encoded by DNA cloned from the mouse.
Populations of Gonyaulax polyedra, in two different phases, about 11 h apart, were mixed, and the intensity of their spontaneous bioluminescence glow recorded for about 2 wk under conditions of constant dim (35 +/- 3 microE/m2/s) white light and constant temperature (19.0 +/- 0.3 degrees C). The phases and amplitudes of glow signals recorded from mixed cultures were compared with those obtained from the arithmetic sum of the intensity data from two control vials. Peaks in control cultures generally remained separate, but there was a spontaneous increase in the period beginning 6-11 d after the onset of constant conditions. This did not occur in cultures in which the medium was exchanged with fresh medium every 2 d. In the actual mixes of two cultures there was a merging of the two subpeaks in the signal, which did not occur when the medium was exchanged. The results indicate that conditioning of the medium by cells may affect the period of the circadian rhythm and that this might result in a type of communication.
The per locus of Drosophila has been implicated in the control of behavioural rhythms. In fruitfly embryos and larvae per is expressed in salivary glands. Per mutations have striking effects on intercellular communication in salivary glands: gap junction channels are modulated so that their conductance varies inversely with the period of behavioural rhythms in the mutants. A similar effect on junctional communication in the nervous system may explain how per influences behavioural rhythms.
The per locus influences biological rhythms in Drosophila melanogaster. In this study, per transcripts and proteins were localized
in situ in pupae and adults. Earlier genetic studies have demonstrated that per expression is required in the brain for circadian
locomotor activity rhythms and in the thorax for ultradian rhythmicity of the Drosophila courtship song. per RNA and proteins
were detected in a restricted group of cells in the eyes and optic lobes of the adult brain and in many cell bodies in the
adult and pupal thoracic ganglia. per products were also found in the pupal ring gland complex, a tissue involved in rhythmic
aspects of Drosophila development. Abundant expression was seen in gonadal tissue. No biological clock phenotypes have been
reported for this tissue in any of the per mutants, per protein mapped to different subcellular locations in different tissues.
The protein accumulated in or around nuclei in some cells and appeared to be cytoplasmic in others.
The endogenous and ubiquitous circadian rhythms (CR) are not understood in molecular terms, in spite of recent interesting advances. They concern, on one hand, protein synthesis, of which a small fraction--possibly a single protein--, formed on cytosolic ribosomes, may be required on each 24 h cycle. On the other hand, the per gene, involved in the control of CR in drosophila has been found to direct the synthesis of 2 (or 3) proteoglycans. Several models have been put forward in order to explain CR generation. Considering the complexity of the cell's organization and the occurrence of partial arrhythmicity, CR generation might result from the integration of a few physiological and metabolic pathways normally involving at least one feed-back loop. Sequentiality would be inherent to the kinetics of both the metabolic pathways and translocators located in the membranes of the various compartments; as a consequence, the peaks of the oscillatory activities would be positioned at particular phase points relative to others. Proteoglycans (or other proteins modified post-transcriptionally) could be involved in the operation of rhythms in controlling not only some plasmalemma (as proposed by Yu et al., 1987) but also intracellular membranes. Finally, reversible enzyme modification occurring on each 24 h cycle could be critically important in circadian rhythms generation.
Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.