Circadian Rhythm of Pattern Formation in Populations of a Free-Swimming Organism,Tetrahymena*

SYNOPSIS. In a preliminary search for a phototactic response in Tetrahymena, we discovered instead an autotactic phenomenon of pattern formation produced by motile free-swimming cells in a dense culture. This pattern of cell aggregation is remarkably similar to the Bénard-cell patterns of classical physical chemistry, and reminiscent of the streaming patterns of motile microorganisms. In a study of light effects on the rate of pattern formation, we found that it has an endogenous circadian component, and is strongly dependent on cell concentration.     

时钟节律与细胞周期

昼夜节律和细胞周期是生命有机体中两种主要的节律性、周期性的活动,参与机体代谢与生理节律．在分子水平上,它们的周期性活动是由一种周期性振荡的网络构成的,这种网络由一系列节律性表达的蛋白所形成．研究发现,多种节律因子通过调节周期蛋白的表达影响细胞周期进程,如G ₁-S期,REV-ERBa抑制p21促进细胞进程,RORα激活p21抑制细胞进程,DEC1抑制cyclinD1,CLOCK/BMAL1负调控c-Myc;G ₂-M期,BMAL1/CLOCK、BMAL1/NPAS2或Cry1作用于Wee1抑制或激活G_2-M期进程．此外,昼夜节律钟蛋白也参与了DNA损伤修复及细胞死亡的过程：Per1、Tim分别作用于ATM、ATR,因而促进细胞周期停滞,p53缺失的细胞中敲除Cry促进细胞凋亡过程,抑制了肿瘤的形成,DEC1以p53依赖的方式促细胞衰老等．同时,节律因子的紊乱引起多种疾病的产生．因此,阐明昼夜节律对细胞周期及死亡的影响,将为肿瘤的治疗提供分子理论基础．     

Synchronization of the Cell Cycle of Tetrahymena by Starvation and Refeeding*

SYNOPSIS The present report describes a simple and useful method for synchronizing mass cultures of the ciliate Tetrahymena pyriformis. The method employs a nutritional approach which involves starvation of the cells in a non-nutrient phosphate buffer followed by refeeding with an enriched nutritional growth medium. It takes 240 minutes after refeeding before the first cells start to divide. Radioautographic and DNA determinations taken together show that starved cells are stalled in the GI nuclear DNA condition and that essentially all of the cells replicate their DNA prior to their first cell division.     

Zebrafish Temperature Selection and Synchronization of Locomotor Activity Circadian Rhythm to Ahemeral Cycles of Light and Temperature

In addition to light cycles, temperature cycles are among the most important synchronizers in nature. Indeed, both clock gene expression and circadian activity rhythms entrain to thermocycles. This study aimed to extend our knowledge of the relative strength of light and temperature as zeitgebers for zebrafish locomotor activity rhythms. When the capacity of a 24∶20°C (thermophase∶cryophase, referred to as TC) thermocycle to synchronize activity rhythms under LL was evaluated, it was found that most groups (78%) synchronized to these conditions. Under LD, when zebrafish were allowed to select the water temperature (24°C vs. 20°C), most fish selected the higher temperature and showed diurnal activity, while a small (25%) percentage of fish that preferred the lower temperature displayed nocturnal activity. Under conflicting LD and TC cycles, fish showed diurnal activity when the zeitgebers were in phase or in antiphase, with a high percentage of activity displayed around dawn and dusk (22% and 34% of the total activity for LD/TC and LD/CT, respectively). Finally, to test the relative strength of each zeitgeber, fish were subjected to ahemeral cycles of light (T=25 h) and temperature (T=23 h). Zebrafish synchronized mostly to the light cycle, although they displayed relative coordination, as their locomotor activity increased when light and thermophase coincided. These findings show that although light is a stronger synchronizer than temperature, TC cycles alone can entrain circadian rhythms and interfere in their light synchronization, suggesting the existence of both light‐ and temperature‐entrainable oscillators that are weakly coupled.     

The Shape of the Endogenous Circadian Rhythm of Rectal Temperature in Humans

Fourteen healthy subjects have been studied in an isolation unit while living on a 30h “day” (20h awake, 10h asleep) for 14 (solar) days but while aware of real time. Waking activities were sedentary and included reading, watching television, and so forth. Throughout, regular recordings of rectal temperature were made, and in a subgroup of 6 subjects, activity was measured by a wrist accelerometer. Temperature data have been subjected to cosinor analysis after “purification,” a method that enables the endogenous (clock-driven) and exogenous (activity-driven) components of the circadian rhythm to be assessed. Moreover, the protocol enables effects due to the circadian rhythm and time-since-waking to be separated. Results showed that activity was slightly affected by the endogenous temperature rhythm. Also, the masking effects on body temperature exerted by the exogenous factors appeared to be less than average in the hours before and just after the peak of the endogenous temperature rhythm. This has the effect of producing a temperature plateau rather than a peak during the daytime. The implications of this for mental performance and sleep initiation are discussed. (Chronobiology International, 13(4), 261-271, 1996)     

The Effects of Temperature and Heavy Water on Cell Division in Heat-Synchronized Cells of Tetrahymena*

SYNOPSIS. A study was undertaken of the rates of cell division of heat-synchronized cells of Tetrahymena pyriformis GL at various temperatures in water and 20, 30 and 40% heavy water. The results suggest that division rate is limited by a protein which undergoes both high and low temperature denaturation and that this protein is partially stabilized against heat-denaturation while becoming more susceptible to cold denaturation in the presence of heavy water. Thus, the optimum temperature for division shifts upward as the heavy water concentration is increased, with a maximum shift of 1 C occurring in 40% heavy water. In addition, division activity occurs in heavy water at 34 C, a temperature at which cells kept in water are blocked. Furthermore, the sharp increase in slope seen in the low temperature portion of Arrhenius plots of the data, occurs at higher temperatures when heavy water is present. Finally, at virtually all temperatures, except the highest, heavy water has a depressive effect on division rate indicating a general inhibitory influence of deuterium-substituted water on rate processes within the cell.     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.     

Absence of Seasonal Variation in the Phase of the Endogenous Circadian Rhythm in Humans

Humans may be subject to seasonal variations, as evidenced by the existence of seasonal affective disorder (SAD) and midwinter insomnia. However, some recent studies have shown that the seasonal variation in the phase of the circadian rhythm is relatively weak in healthy humans. In the present study, evidence is found that there is no seasonal variation in the phase of the endogenous circadian rhythm at all. Body temperature, cortisol excretion, and subjective alertness of six subjects recorded under constant routine conditions showed no systematic seasonal variation in circadian phases. This finding indicates that secondary zeitgebers blocked or counterbalanced the seasonal variation in the entrainment effect of the natural photo-period. The human being may live in an environment in which the photoperiod has lost its status of primary zeitgeber. (Chronobiology International, 15(6), 623-632, 1998)     

Frequency Resonances of the Circadian Rhythm of CAM Under External Temperature Rhythms of Varied Period Lengths in Continuous Light*

Net CO₂ exchange (J_co2) of leaves of the CAM plant Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie was studied in continuous light under the influence of temperature jumps of different period lengths. Each period consisted of two equal time intervals at a lower and a higher temperature, i.e. ?24 and ?29 °C respectively. At a period length of the imposed external temperature rhythm of 8 h the free running endogenous rhythm of J_co2 with a period length of 21.7 h was expressed. Under external rhythms of 12–28 h period length there was entrainment of J_co2. Under external rhythms of 40 and 56 h the time course of J_co2 became arrhythmic. In power spectra of the J_co2 time series obtained by Fourier analysis, besides the dominant frequency, there were other frequency peaks with lower Fourier amplitudes of J_co2. It was noted that these are natural number multiples of the major frequencies. A comparison shows that these frequencies correspond to the harmonic overtones of the basic oscillation of the free running endogenous rhythm of J_co2 at 24°C, viz. 1/22.5 h^?1. The overall power of the enforced oscillations shows a typical resonance behaviour around the natural, free running frequency. The intriguing observation that external temperature jump rhythms lead to resonance, not only of the basic oscillation but also of its harmonic overtones, is discussed in relation to earlier findings suggesting the occurrence of paths between order and deterministic chaos in the CAM rhythm.     

Synchronization of Chromosome Dynamics and Cell Division in Bacteria

Bacterial cells have evolved a variety of regulatory circuits that tightly synchronize their chromosome replication and cell division cycles, thereby ensuring faithful transmission of genetic information to their offspring. Complex multicomponent signaling cascades are used to monitor the progress of cytokinesis and couple replication initiation to the separation of the two daughter cells. Moreover, the cell-division apparatus actively participates in chromosome partitioning and, particularly, in the resolution of topological problems that impede the segregation process, thus coordinating chromosome dynamics with cell constriction. Finally, bacteria have developed mechanisms that harness the cell-cycle-dependent positioning of individual chromosomal loci or the nucleoid to define the cell-division site and control the timing of divisome assembly. Each of these systems manages to integrate a complex set of spatial and temporal cues to regulate and execute critical steps in the bacterial cell cycle.In recent years, considerable progress has been made in understanding the cell biology of bacteria and, in particular, the organization and dynamics of their chromosomes (Shih and Rothfield 2006; Graumann 2007; Morris and Jensen 2008; Reyes-Lamothe et al. 2008b; Thanbichler and Shapiro 2008). It has emerged that bacterial cells have evolved a variety of mechanisms to closely coordinate replication and segregation of chromosomal DNA with cell division, thus ensuring that genetic information is passed on faithfully. In the absence of these regulatory circuits, premature formation of a division septum can lead to dissection of the nucleoid and generation of anucleate cells. Conversely, untimely origin firing before the end of cell division may result in the accumulation of supernumerary chromosomes, thereby interfering with proper cell cycle and gene regulation, and loading the cell with a substantial metabolic burden. This article first summarizes our current knowledge on checkpoints that couple replication initiation and the last stages of chromosome segregation to the progression of cell constriction. Subsequently, it discusses effects of chromosome dynamics on the temporal and spatial control of divisome assembly.     

Circadian Rhythm Changes in Autotrophic Euglena Induced by Organic Carbon Sources*

SYNOPSIS. Acetate added to autotrophic Euglena cultures changed the period length of the circadian rhythm of phototaxis. Phase shifts were induced by acetate pulses. Since transition from one metabolic state to another (autotrophic/mixotrophic) caused a phase shift or a period change, such effects possibly result from switching metabolic pathways. As suggested (Brinkmann, K., 1966. Planta 70 , 344–89), differences in the temperature responses of the rhythm in mixotrophic and autotrophic cells might also be caused by participation of different metabolic pathways with different Q¹⁰ values, e.g. dark reactions vs photochemical reactions. However the Q¹⁰ of a given dark reaction, e.g. protein synthesis, can differ in the 2 states. Therefore temperature experiments alone do not suffice for deciding whether the pathways include photochemical reactions, dark reactions, or both.     

The Action of Mercaptoethanol on Cell Division in Synchronized Tetrahymena1

SYNOPSIS. Action of mercaptoethanol (ME) on cell division and macromolecular synthesis was examined in Tetrahymena synchronized for division. Cells continuously exposed to increasingly higher concentrations of ME divided with progressively longer division delays showing a dosage-dependent response to the agent. Division was blocked in 2 × 10^?2 M ME. Many cells cytolyzed in high concentrations of ME (4 × 10^?2 M); others became spherical and motility decreased. Non-delaying concentrations of ME (2 × 10^?2 M) had little or no effect on protein synthesis but decreased DNA and RNA synthesis 10 and 35%, respectively. Blocking concentrations inhibited incorporation of phenylalanine, thymidine and uridine 35, 60, and 85%, respectively. It is suggested that the mode of action of ME is mediated thru inhibition of macromolecular synthesis essential for cell division and thru inhibition of formation of disulfide bridges between protein subunits.     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.     

Partial Synchronization of Cell Division in Suspension Cultures of Haplopappus gracilis

Four different chemicals were tested in their ability to synchronize cell division in asynchronous cell cultures of Haplopappus gracilis. Twentyfour-hour treatments with 5-amino uracil resulted in a peak in the mitotic index about 14–16 hours after the end of the treatment. The increase in the frequency of mitoses was about three times that of the control. Hydroxyurea, at a concentration of 3 mM, gave after a treatment period of 12–24 hours an increase in the frequency of mitoses which appeared about 10 hours after the treatment. The mitotic index was about 35 per cent, which is 4 times that of the control. 5-Fluorodeoxyuridine (FUdR) at a concentration of 2 × 10^?7M gave a mitotic burst about 16 hours after treatment. At that time about 15 per cent of the cells were dividing which was about twice that of the control. The block was reversed with 4 × 10^?5M thymidine. Thymidine at a high concentration caused a reduction in the frequency of mitoses during the treatment. After 15 to 16 hours in a thymidine free medium a mitotic peak appeared with a doubling of the frequency of mitoses in treated cells. Cytological studies showed that parlicularly hydroxyurea but also 5-aminouracil and 5-fluorodeoxyuridine produced gaps and fragments at the concentrations which gave cell synchronization.     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Circadian Rhythm in Pineal Methionine S-Adenosyltransferase

Abstract: The circadian rhythm of methionine S -adenosyltransferase, which catalyzes the formation of S -adenosylmethionine, a cosubstrate for melatonin in the pineal gland, follows the pattern of hydroxyindole- O -methyltransferase. Around the middle of the dark period, methionine S -adenosyltransferase and hydroxyindole- O -methyltransferase appear to be elevated by 2.5- and 1.5-fold, respectively, and tend to fall back during the light period.     

Circadian Rhythm in Serum Iron Levels

This study was aimed at assessing the circadian rhythm of serum iron levels in Chinese healthy subjects. The project was conducted in 19 healthy, Chinese male subjects following a 4-day diet equilibration. Blood samples were collected on day 5 at 0800, 1000, 1200, 1400, 1600, 1800, 2000, and 2400 hours to determine endogenous serum iron concentrations. Iron concentrations were determined using an inductively coupled plasma mass spectrometry. Iron concentration was decreased from morning to afternoon. The minimum value of iron level was 1,987 μg/L at 2000 hours while the maximum was 2,229 μg/L at 1000 hours, and 2,278 μg/L at 1400 hours, respectively, the amplitude was 291 μg/L. This study indicates that when assessing the bioequivalence of iron formulations, baseline levels of iron (obtained before dosing) should not be subtracted simply from the amount obtained on the drug dosing day to yield the net effect of iron formulation administration. More valid methods to optimize the design of such bioequivalence studies should be taken into consideration.