The in Vitro Effect of Metyrapone on Steroid Synthesis in Mice Adrenals at Different Circadian Stages

The circadian rhythm of the in vitro biosynthesis of Cortisol and cortisone in mice adrenals has been documented in the absence and presence of 0.1 μmol metyrapone, an inhibitor of steroid 11β-monooxygenase. After 3 weeks of synchronization with 12 h light: 12 h darkness, adrenalectomy was performed at eight circadian stages: 0, 4, 9, 10, 13, 16, 21, and 22 h after light onset (HALO). Because it has been shown that mice adrenals could convert exogenous 11 -deoxy-cortisol, the synthesis of 11-oxysteroids (Cortisol + cortisone) in adrenal homoge-nates was studied from tritiated precursor. The pattern of steroid synthesis showed a maximum around the end (10 HALO) and a minimum at the beginning of the resting period (0 HALO); the variation was ～ 10%. A similar pattern was observed in the presence of a ～50% inhibiting dose of metyrapone. On the other hand, the percent inhibition of 11-oxysteroids synthesis was greater at the beginning of the resting period (0 HALO) and minimum around the end of the activity span (21 HALO), with an overall variation of 20%. However, the variations were statistically insignificant (unpaired t test).     

The Use of Salivary Steroids to Monitor Circadian Rhythmicity on Expeditions in the Arctic

The circadian variation in salivary Cortisol concentration was investigated in four men during a traverse on foot, of the Greenland ice cap and of salivary Cortisol and testosterone on a summer expedition to Spitzbergen. In both instances circadian rhythmicity was demonstrated both before leaving the U.K. and throughout the expedition. The acrophase of the rhythms followed the changes in activity patterns on both expeditions although there was a dissociation between the Cortisol and testosterone following an acute 8 hr phase shift in Spitzbergen. The mesor of Cortisol in both instances tended to be higher than in the U.K. but the difference was never statistically significant. This work demonstrates the feasibility of using salivary steroid concentrations to investigate their circadian rhythmicity in circumstances where frequent blood sampling would be precluded.     

Gene-Dependent Effect of Lithium on Circadian Rhythms in Mice (Mus Musculus)

Lithium has been shown to lengthen free-running circadian periods in a variety of species. Here we show that lithium carbonate differentially lengthens the free-running period of a circadian wheel running rhythm in BALB/CByJ and C57BL/10Sn inbred mouse strains. This result supports previous evidence that lithium lengthens mammalian circadian rhythms, and also demonstrates that gene differences can mediate individual differences in response to lithium treatment.     

Influence of Circadian Time Structure on Acute Hormonal Responses to a Single Bout of Heavy-Resistance Exercise in Weight-Trained Men

Both testosterone (T) and cortisol (C) exhibit circadian rhythmicity being highest in the morning and lowest in the evening. T is a potent stimulator of protein synthesis and may possess anti-catabolic properties within skeletal muscle, and C affects protein turnover, thereby altering the balance between hormone-mediated anabolic and catabolic activity. Physiological reactions of these hormones and training adaptations may influence the post-exercise recovery phase by modulating anabolic and catabolic processes, therefore affecting metabolic equilibrium, and may lead to intensification of catabolic processes. We investigated the effect of the circadian system on the T and C response of weight-trained men to heavy resistance exercise. Thirteen young (21.8±2.2 yr) weight-trained men (12 months training experience) performed an eight-station heavy-resistance exercise protocol on two separate occasions (AM: 06:00 h and PM: 18:00 h), completing 3 sets of 8–10 repetitions at 75% of each subject's one-repetition maximum (1-RM). Blood samples were obtained prior to, during, and following the exercise bout, and serum total T and C concentrations were determined by competitive immunoassay technique. Performing the single bout of heavy-resistance exercise in the PM as compared to the AM positively altered the C and T/C ratio hormonal response. Pre-exercise C concentrations were significantly lower (p < 0.05) in the PM session, which resulted in a lower peak value, and the accompanying increased T/C ratio suggested a reduced catabolic environment. These data demonstrate that the exercise-induced hormonal profile can be influenced by the circadian time structure toward a profile more favorable for anabolism, therefore optimizing skeletal muscle hypertrophic adaptations associated with resistance exercise.     

Circadian Modification of 5-Fluorouracil-Induced Teratogenesis in Mice

Pregnant mice were treated intraperitoneally with 5-fluorouracil in a single dose of 30 mg/kg once on gestation day 11 at one of four selected times along the 24-hr time scale. All animals were kept under a lighting regimen of 12 hr (0600-1800) alternating with 12 hr of darkness. They were injected at the same circadian phase as they were mated. The developmental age of all fetuses was 264 ± 1 hr at the time of injection. Fetuses collected on day 18 showed a circadian variation in teratogenic susceptibility to 5-fluorouracil. The lethal and teratogenic risk posed by the drug were highest among animals treated at the mid-light period until the onset of darkness.     

The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.     

Circadian Rhythm of Irinotecan Tolerability in Mice

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12 h light/12 h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO ( p = 0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage ( p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals’ rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.     

Effect of Colostomy on the Circadian Rhythm in DNA Synthesis in the Rat Colon

Synthesis of DNA and mitosis in gut epithelium are not constant or random events but rather are characterized by circadian rhythmicity, which we reported persists even in fasted rats. Others suggest that rhythms persist because rats anticipate food, causing nerve impulses to propagate caudally in the gut at usual meal times, or that digestive products from previous feedings cause rhythms in the lower tract. We studied colonic DNA synthesis in rats that had been given colostomies. In one study, the distal colon was isolated neurally from proximal gut by means of an end colostomy. In a second study, rats were subjected to loop colostomy; some intrinsic innervation of the gut wall remained intact. Sprague-Dawley male rats, 8 weeks old, were acclimated to a 12:12 light-dark cycle. Colostomies were performed after a 48-h fast. The rats were fed ad libitum for 4 weeks after surgery. Operated rats and an equal number (n = 30) of control rats (unoper-ated) were divided into four subgroups that were killed at 07:00,13:00,19:00, and 01:00 h. Each rat was injected with tritiated thymidine 30 min before it was killed. Proximal and distal colon were analyzed for incorporation of radioactivity (DNA synthesis). Results are reported as counts per minute per microgram of DNA and were analyzed using analysis of variance and the t test. Significant daily variation was found in proximal colon, both from control and operated rats. Rhythms were still present in colon distal to loop colostomy but were lost in the distal stump in rats that received an end colostomy. The mammalian “biological clock” regulates most circadian rhythms by neurohumoral mechanisms; however, our results suggest that the intrinsic enteric nervous system is important in the propagation of a signal that causes rhythmic cell proliferation in the gut in the intact animal.     

Circadian rhythm characteristics of serum cortisol and dehydroepiandrosterone sulfate in healthy Chinese men aged 30 to 60 years. A cross-sectional study

The relation of adrenal function and aging has been the subject of intense interest in recent years. The circadian variations of plasma cortisol have been described in Caucasians, but little information is available on such hormone variations among the Chinese population, especially its changes with age. This study was, therefore, designed to examine the effects of age on the circadian variations of serum cortisol, dehydroepiandrosterone sulfate (DHEAS) and the molar ratio of cortisol/DHEAS in Chinese men, stratified by 10-year age-groups (i.e. men in their 30-60s, aged from 31 to 63 years old). Circadian variations of serum cortisol and DHEAS were documented at 2-h intervals from 8:00 to 22:00 and hourly from 22:00 to 8:00 in 26 healthy Chinese men. We found that the serum levels of both hormones showed a statistically significant circadian rhythmicity in all age-groups. The circadian pattern of serum cortisol was characterized by peaks (04:00-06:00) and troughs (18:00-24:00) occurring approximately 2h earlier than those usually reported in Caucasians. Aging did not significantly influence serum cortisol concentrations, but serum DHEAS levels declined significantly with age: subjects in their 60s had significantly lower levels, and their cortisol/DHEAS molar ratios were significantly higher than those in the younger age-groups.     

Plasticity of Circadian Activity and Body Temperature Rhythms in Golden Spiny Mice

Most animals can be categorized as nocturnal, diurnal, or crepuscular. However, rhythms can be quite plastic in some species and vary from one individual to another within a species. In the golden spiny mouse (Acomys russatus), a variety of rhythm patterns have been seen, and these patterns can change considerably as animals are transferred from the field into the laboratory. We previously suggested that these animals may have a circadian time‐keeping system that is fundamentally nocturnal and that diurnal patterns seen in their natural habitat reflect mechanisms operating outside of the basic circadian time‐keeping system (i.e., masking). In the current study, we further characterized plasticity evident in the daily rhythms of golden spiny mice by measuring effects of lighting conditions and access to a running wheel on rhythms in general activity (GA) and body temperature (Tb). Before the wheel was introduced, most animals were active mainly during the night, though there was considerable inter‐individual variability and patterns were quite plastic. The introduction of the wheel caused an increase in the level of nighttime activity and Tb in most individuals. The periods of the rhythms in constant darkness (DD) were very similar, and even slightly longer in this study (24.1±0.2 h) than in an earlier one in which animals had not been provided with running wheels. We found no correlation between the distance animals ran in their wheels and the period of their rhythms in DD. Re‐entrainment after phase delays of the LD cycle occurred more rapidly in the presence than absence of the running wheel. The characteristics of the rhythms of golden spiny mice seen in this study may be the product of natural selection favoring plasticity of the circadian system, perhaps reflecting what can happen during an evolutionary transition as animals move from a nocturnal to a diurnal niche.     

Provisional QTL for Circadian Period of Wheel Running in Laboratory Mice: Quantitative Genetics of Period in RI Mice

Wheel running was monitored in B X D recombinant inbred (RI) mice under dark-dark (DD) conditions, and the mean circadian period was calculated for each strain. There were significant differences for this trait among B X D recombinant inbred strains (p <. 0001) and a narrow-sense heritability of 21%. Analysis of strain means and variances indicates that at least four segregating loci contribute to the genetic variance for the free-running circadian period in this population. Correlation of the strain means for the circadian period of wheel running for each RI strain against the distribution of markers at over 1500 loci along the mouse genome identified a number of provisional quantitative trait loci (QTL). There were provisional QTL for wheel running atp <. 001 on chromosome 11 and atp <. 01 on chromosomes 1, 6, 9, 17, and 19. Most were in agreement with a second analysis done under similar conditions.     

Circadian Rhythms in Toxic Effects of the Serotonin Antagonist Ondansetron in Mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren®) administration is dosing‐time dependent. Ondansetron is a serotonin 5‐HT₃ receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo‐ and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12h light (rest span)/12h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD₅₀) and lethal (LD₅₀) doses, which were, respectively, 3.7 ± 0.6 mg/kg and 4.6 ± 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time‐dependent (χ² = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one‐half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (χ² = 22.24, p < 0.0001). Effects on rectal temperature were also dosing‐time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (τ ≡ 8h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24h and 8h periodicities.     

Circadian Variation in Alloxan Sensitivity of Mice as Indicated by Mortality and Blood Glucose Alteration

The objective of this study was to determine in mice if a time-dependent pancreatic β-cell susceptibility to alloxan could be correlated to daily changes in blood glucose levels and to monitor the pattern of blood glucose at various times of day as mice became diabetic. Food was removed from mice standardized to a 12-h light:dark cycle (lights on at 0600 h CST, during the month of June) at 12 h before subcutaneous injection with 0.27 mg/g of alloxan. Six groups of 30 fasted mice were injected at 4-h intervals. Blood glucose levels were measured from each group immediately prior to injection, and at 2, 4, 8, 12, 24, 48, and 216 h after treatment. Animals receiving alloxan during the early- to middark period had an increase in blood glucose after 2 h, followed by a decline to hypoglycemic levels between 4 and 8 h, and recovery to hyperglycemic levels 48 h after alloxan exposure. Three and 30% of these animals were dead at 8 and 48 h, respectively. Mice treated during the midlight span had decreased blood glucose levels 2 h after alloxan treatment followed by an increase to diabetic hyperglycemia within 48 h. Twenty-three and 70% of the animals treated at 1430 h were dead at 8 and 48 h, respectively. At 216 h, total mortality was 45.6% and 81 of the 98 surviving mice were hyperglycemic. These data suggest a greater sensitivity to alloxan during the midlight resting period of the mice. This may be the result of increased sensitivity to the insulin released from the β cells when alloxan was given during the light span.     

Short-Day Response in Djungarian Hamsters of Different Circadian Phenotypes

In Djungarian hamsters (Phodopus sungorus) bred at the authors' institute, a certain number of animals show activity patterns incompatible with proper entrainment of their endogenous circadian pacemaker to the environmental light-dark (LD) cycle. Even though the activity-offset in these animals is stably coupled to “light-on,” activity-onset is increasingly delayed, leading to a compression of the activity time (α). If α falls below a critical value, the circadian rhythm in these so called delayed activity-onset (DAO) hamsters starts to free-run and finally breaks down. Animals then show an arrhythmic activity pattern (AR hamsters). Previous studies revealed the mechanisms of photic entrainment have deteriorated (DAO) or the suprachiasmatic nucleus (SCN) does not generate a rhythmic signal (AR). The aim of the present study was to investigate the consequences that these deteriorations have upon photoperiodic time measurement. Animals were bred and kept under standardized housing conditions with food and water ad libitum and a 14L/10D (long day, LD) regimen. Locomotor activity was recorded continuously using passive infrared motion detectors. Body mass, testes size, and fur coloration were measured weekly or biweekly to further quantify the photoperiodic reaction. In a first experiment, adult male wild-type (WT), DAO, and AR hamsters were transferred initially to a 16L/8D cycle. After 3–4 wks, the light period was shortened symmetrically by 8?h. After 14 wks, none of the DAO and AR hamsters, and only 1 of 8 WT hamsters showed short-day (SD) traits. Therefore, in a second experiment, hamsters were transferred to SD conditions (8L/16D cycle) for 8 wks directly from standard LD conditions. In 6 of 7 WT hamsters, activity time expanded, body mass and testes size decreased, and fur coloration changed from summer to winter pelage. In contrast, none of the DAO and AR hamsters displayed an SD response. In a third experiment, DAO and AR hamsters were kept in constant darkness (DD) for 8 and 14 wks. After 8 wks, DAO hamsters showed a similar photoperiodic reaction to WT hamsters that had been kept for 8 wks under SD conditions. However, the level of adaptation was still less compared to WT hamsters, but this difference was not apparent after 14 wks. In contrast, AR animals did not display any photoperiodic reaction, even after 14 wks in DD. Type VI phase response curves (PRCs) were constructed to better understand the mechanism behind the SD response. In WT hamsters, the photosensitive phase, where light pulses induce phase shifts, was lengthened in SD condition. In DAO hamsters, in contrast, the PRCs were similar under LD and SD conditions with a compressed photosensitive phase corresponding to α. Also, “light-on” induced only weak phase advances of activity-onset, insufficient to compensate for the long endogenous period. The results show that physiological mechanisms necessary for seasonal adaptation are working in DAO hamsters and that it is the inadequate interaction of the LD cycle with the SCN that prevents the photoperiodic reaction. AR hamsters, on the other hand, are incapable of measuring photoperiodic time due to a complete disruption of circadian rhythmicity.     

Circadian and Acamprosate Modulation of Elevated Ethanol Drinking in mPer2 Clock Gene Mutant Mice

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12?h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2?h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ～60 vs. ～25?mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300?mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout. (Author correspondence: jglass@kent.edu)     

Effect of Ethanol and Theophylline on Circadian Rhythm of Rat Locomotion

The effect of ethanol and theophylline on the circadian rhythm of rat locomotion was investigated. Male Wistar rats synchronized to 12: 12 h light-dark cycles were divided into four groups for treatment with saline, ethanol, theophylline, and ethanol plus theophylline. Animals in each group were orally administered saline, ethanol (2.0 g/kg body wt), theophylline (10 mg/kg body wt), and ethanol plus theophylline, respectively, six times every 2 h during the 12-h light span. Spontaneous loco-motor activity was continuously monitored by an animal activity recorder at 15-min intervals. Total activity count, circadian rhythm characteristics of activity (amplitude, acrophase, and mesor), power spectral patterns, and slope of fluctuation (a measurement of ultradian periodicity) were calculated. Ethanol administration decreased the total activity count by 60% and phase-delayed the onset of activity rhythm by 9.5 h on the day after treatment. The absolute value of the slope of fluctuation was increased by ethanol administration. The mean recovery time evaluated by rhythm detection was 3.8 days. Theophylline administration increased the light phase activity, but caused no phase delay of the onset time of the locomotor activity rhythm. The decrease in total activity count and phase delay of onset of the activity rhythm caused by ethanol were partially antagonized by theophylline. However, the prolonged effects of ethanol, represented by a late recovery time and an increase in the slope of fluctuation, were not influenced by theophylline.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythm of Total Protein Synthesis in the Cytoplasm and Chloroplasts of Gonyaulaxpolyedra

Protein synthesis of Gonyaulax polyedra was analyzed by means of electron microscopic autoradiographs under constant conditions at different times of the 24-hr cycle. Circadian rhythmic changes in the synthesis rate of total protein were determined in the cytoplasm and chloroplasts of growing cells. Three independent series of experiments in constant light showed a maximum of grains per unit area during the 'subjective' dark phase (=phase that corresponds to the dark phase during a 12:12 hr LD cycle) in both compartments. Minimum and maximum grain number are different by a factor of 5-10. The maximum of total protein synthesis coincided with the maximum phase shift by cycloheximide pulses (1) suggesting protein species within the total pool involved in the mechanism of the circadian clock. A similar rhythm of lower amplitude was observed in the mitochondria, but this rhythm cannot with certainty be attributed to these organelles. In a slowly growing culture a rhythm of total protein synthesis was observed that showed a smaller amplitude and a different phasing.