Accidents in children do not happen at random: Predictable time-of-day incidence of childhood trauma

In a prospective study, 15,110 childhood traumas were recorded by the Pediatric Surgery Service (CHUV, Lausanne) between January 1, 1990 and December 31, 1997. The exact clock hour when the injury occurred and other germane data were obtained. Time series thus obtained were analyzed by several statistical (ANOVA, cosinor, χ², Table Curve, etc.) methods. High statistically significant circadian patterns were detected with a trough at night—almost no traumas/hour (t/h), and a peak in the afternoon (～16:00h)—9.3±0.4 (SD) t/h. Such 24h variation was validated for the whole sample for the entire 8yr study span as well as the data of each year. Neither gender- nor age-related differences in the 24h pattern were detected between children under 5 yr of age, who have not yet attended school and children from 5 to 16 yr of age, who attend school. Small but statistically significant differences in the 24h patterns were observed when categorized by the type of activity associated with the trauma and the place of trauma occurrence. The great stability of the 24h pattern in childhood trauma over the 8yr study span suggests an endogenous origin in addition to the role presumably played by environmental factors. Periods of 12 and 8 h were also detected in the time series. The afternoon peak time of childhood traumas differs from that of adults, which is located ～04:00h in rotating shift workers and automobile drivers and 06:00–08:00h in adult day-workers. The validation of a circadian pattern in childhood traumas with an afternoon peak should be taken into account in the design of children's preventative injury programs.     

Reproductive phase dependent circadian variations of plasma melatonin, testosterone, thyroxine and corticosterone in Indian palm squirrel, Funambulus pennanti

To explain the complex mechanism of environmental influence along with internal hormonal (factors) milieu on daily variations in the circulating levels of melatonin, testosterone, thyroxine and corticosterone were analyzed with the help of inferential statistics (Cosinor rhythmometry) in a seasonally breeding tropical rodent, F. pennanti during the reproductively active (RAP) and inactive phases (RIP). Plasma melatonin, thyroxine and corticosterone levels exhibited a significant circadian oscillation during both the active and inactive phases of the annual reproductive cycle. Melatonin showed higher amplitude during RIP in the circulating plasma. Testosterone presented a peak level during evening hours (16:00 - 18:00 h) during RAP only. The phase of thyroxine was noted ∼09:76 h and ∼10.35 h during active and inactive phases, respectively. Corticosterone showed a peak level at ∼12.00 h during both phases of the reproductive cycle. Further, in this tropical rodent, the minimum difference in photoperiod (∼3 - 4 hours) and maximum variation in temperature (max. 18°C - min. 10°C during RIP and max. 45°C - min. 32°C during RAP) and humidity (85% during RIP and 35% during RAP) regulated the diurnal rhythm of circulating melatonin circadian rhythm by ∼1 hour phase advance during RIP. In conclusion, the studied hormonal rhythms may be part of an integrative system to coordinate reproduction and physiological processes successfully with environmental factors.     

Circadian variation in oxidative stress markers in healthy and type II diabetic men

Seven clinically healthy, nondiabetic (ND) and four Type II diabetic (D) men were assessed for circadian rhythms in oxidative “stress markers.” Blood samples were collected at 3h intervals for ∼27 h beginning at 19:00h. Urine samples were collected every 3 h beginning with the 16:00h-19:00h sample. The dark (sleep) phase of the light-dark cycle extended from 22:30h to 06:30h, with brief awakening for sampling at 01:00h and 04:00h. Subjects were offered general hospital meals at 16:30h, 07:30h, and 13:30h (2400 cal in total/24 h). Serum samples were analyzed for uric acid (UA) and nitrite (NO) concentrations, and urine samples were assayed for 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), and 8-isoprostane (ISP). Data were analyzed statistically both by the population multiple-components method and by the analysis of variance (ANOVA). The 24h mean level of UA and NO was greater in D than in ND subjects (424 vs. 338 μmol/L and 39.2 vs. 12.7 μM, respectively). A significant circadian rhythm in UA (p=0.001) and NO (p=0.048) was evident in ND but not in D (p=0.214 and 0.065). A circadian rhythm (p=0.004, amplitude=8.6 pmol/kgbw/3h urine vol.) was also evident in urine 8-OHdG of ND but not of D. The 24h mean levels of ND and D were comparable (76.8 vs. 65.7 pmol/kgbw/3h urine vol.). No circadian rhythm by population multiple-components was evident in MDA and ISP levels of ND subjects, or in 8-OHdG, MDA, and ISP in D. However, a significant time-effect was demonstrated by ANOVA in all variables and groups. The 24h mean of MDA and ISP in D was significantly greater than in ND (214 vs. 119 nmol/3h urine vol. and 622 vs. 465 ng/3h urine vol.). The peak concentrations of the three oxidative “stress markers” in urine, like those of serum NO, occurred early in the evening in both groups of men. This observation suggests a correlation between increased oxidative damage and increased rate of anabolic-catabolic events as evidenced by similarities in the timing of peak NO production and in parameters relevant to metabolic functions.     

Circadian and ultradian (12 h) rhythms of hepatic thiosulfate sulfurtransferase (rhodanese) activity in mice during the first two months of life

Thiosulfate sulfurtransferase (TST) is an important 'enzyme of protection,' that accelerates the detoxification of cyanide, converting it into thiocyanate. The TST physiological rhythm was investigated at wks 2, 4, and 8 of post-natal development (PND) in the mouse. The results revealed a statistically significant gender-related difference, with the highest activity in females, at all the documented PND stages. In the second week of PND (pre-weaning time), the circadian rhythm of the enzyme activity was associated with ultradian components. The prominent circadian rhythm (τ=24 h) peaked at the beginning of the light span, more precisely ∼3 HALO (Hours After Light Onset). A week after weaning (wk 4 of PND), an impairment of the rhythm, with the peak shifted toward the second half of photophase, was recorded. Four to 6 wks later, about wk 8 of PND, the circadian rhythm pattern was stabilized, with its peak then located at the beginning of the dark span (13 HALO). The obtained results showed a 12 h phase-shift of the circadian TST peak time during PND, suggesting that the rhythm stabilization is age-dependent.     

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12 h of light and 12 h of darkness (LD12:12) for at least 3 wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of ∼2×2 mm²). Ten days later, a tumor sample (area of ∼5 mm²) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G₁, S or G₂-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G₂-M phase cells. The approximate average distribution patterns of G₁ and G₂-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G₁, 21 HALO for S and 15 HALO for G₂-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.     

Effects of photophase and altitude on oviposition rhythm of the himalayan strains of Drosophila ananassae

The effects of varying photophase and altitude of origin on the phase angle difference (Ψ) of the circadian rhythm of oviposition during entrainment to light-dark (LD) cycles and the aftereffects of such photophases on the period of the free-running rhythm (τ) in constant darkness (DD) were evaluated in two Himalayan strains of Drosophila ananassae, the high-altitude (HA) strain from Badrinath (5,123 m above sea level=ASL) and the low-altitude (LA) strain from Firozpur (179 m ASL). The Ψ (i.e., the hours from lights-on of the LD cycle to oviposition median) of both strains was determined in LD cycles in which the photophase at 100 lux varied from 6 to 18 h/24 h. The HA strain was entrained by all LD cycles except the one with 6 h photophase in which it was weakly rhythmic, but the LA strain was entrained by only three LD cycles with photophases of 10, 12, and 14 h, but photophases of 6, 8, 16, and 18 h rendered it arrhythmic. Lights-off transition of LD cycles was the phase-determining signal for both strains as oviposition medians of the HA strain occurred∼6 h prior to lights-off, while those of the LA strain occurred∼1 h after lights-off. The Ψ of the HA strain increased from∼2 h in 8 h photophase to∼11 h in 18 h photophase, while that of the LA strain increased from∼11 h in 10 h photophase to∼15 h in 14 h photophase. The aftereffects of photophase of the prior entraining LD cycles on τ in DD were determined by transferring flies from LD cycles to DD. The τ of the HA strain increased from∼19 to∼25 h when transferred to DD from LD 8:16 and LD 18:6 cycles, respectively, whereas the τ of the LA strain increased from∼26 to∼28 h when transferred to DD from LD 10:14 and LD 14:10 cycles, respectively. Thus, these results demonstrate that the photophases of entraining LD cycles and the altitude of origin affected several parameters of entrainment and the period of the free-running rhythm of these strains.     

Controlled exposure to light and darkness realigns the salivary cortisol rhythm in night shift workers

The efficacy of a light/darkness intervention designed to promote circadian adaptation to night shift work was tested in this combined field and laboratory study. Six full-time night shift workers (mean age ± SD:37.1 ± 8.1 yrs) were provided an intervention consisting of an intermittent exposure to full-spectrum bright white light (∼2000 lux) in the first 6 h of their 8 h shift, shielding from morning light by tinted lenses (neutral gray density, 15% visual light transmission), and regular sleep/darkness episodes in darkened quarters beginning 2 h after the end of each shift. Five control group workers (41.1 ± 9.9 yrs) were observed in the presence of a regular sleep/darkness schedule only. Constant routines (CR) performed before and after a sequence of ∼12 night shifts over 3 weeks revealed that treatment group workers displayed significant shifts in the time of peak cortisol expression and realignment of the rhythm with the night-oriented schedule. Smaller phase shifts, suggesting an incomplete adaptation to the shift work schedule, were observed in the control group. Our observations support the careful control of the pattern of light and darkness exposure for the adaptation of physiological rhythms to night shift work.     

Effects of a five-hour exposure of arm and leg to a slightly cool thermal environment in the afternoon on the body temperature rhythm

The purpose of this study was to determine whether the different thermal conditions of the arm and leg due to wearing different types of clothing during the afternoon could modulate the circadian rhythm of body temperature and subjective sleep quality. Six healthy female volunteers were studied twice with two types of clothing, leaving the arm and leg covered or uncovered. The environmental chamber was controlled at 24 ± 0.5°C and 50 ± 5% RH during wakefulness and 28 ± 0.5°C and 50 ± 5% RH during night sleep. One type of clothing consisted of long-sleeved shirts and full-length trousers (Type L, 989 g, 0.991 clo); the other type was of half-sleeved shirts and knee-length trousers (Type H, 750 g, 0.747 clo). One testing session lasted for 16 h from 14:00 to 06:00 h the next morning. Subjects wore Type L or Type H clothing during the afternoon exposure (14:00 - 19:00), and Type L clothing during the evening (19:00 - 22:30 h) and the night sleep (22:30 - 06:00 h). Results were as follows. (1) When wearing Type H rather than Type L clothing, skin temperatures of the arm and leg were significantly lower during the time of exposure, and increased more after the evening. (2) Rectal temperature was not significantly different between the two types of clothing except during the early part of the exposure period, but it decreased during the evening by a significantly greater amount when wearing Type H clothing. (3) Subjective sleep quality was not significantly different between the two clothing types. These results suggest that afternoon exposure of the arm and leg to a slightly cool environment does not have a strong after-effect on the body temperature and subjective sleep quality.     

Sleepiness and sleep in a simulated "six hours on/six hours off" sea watch system

Ships are operated around the clock using rapidly rotating shift schedules called sea watch systems. Sea watch systems may cause fatigue, in the same way as other irregular working time arrangements. The present study investigated subjective sleepiness and sleep duration in connection with a 6 h on/6 h off duty system. The study was performed in a bridge simulator, very similar to those found on ships. Twelve officers divided into two groups participated in the study that lasted 66 h. Half of the subjects started with the 06:00-12:00 h watch and the other half with the 12:00-18:00 h watch. The subjects alternated between off-duty and on-duty for the remainder of the experimental period. Approximately halfway through the experiment, the 12:00-18:00 h watch was divided into two 3 h watches/off-duty periods. The effect of this was to reverse the on-duty/off-duty pattern between the two groups. This enabled all subjects to work the four possible watches (00:00-06:00 h, 06:00-12:00 h, 12:00-18:00 h, and 18:00-24:00 h) in an order that was essentially counterbalanced between groups. Ratings of sleepiness (Karolinska Sleepiness Scale; KSS) were obtained every 30 min during on-duty periods and if subjects were awake during off-duty periods. The subjectively rated duration of sleep was recorded after each off-duty period that preceded watch periods when KSS was rated. The results showed that the average level of sleepiness was significantly higher during the 00:00-06:00 h watch compared to the 12:00-18:00 h and 18:00-24:00 h watches, but not to the 06:00-12:00 h watch. Sleepiness also progressed significantly from the start toward the end of each watch, with the exception of the 06:00-12:00 h watch, when levels remained approximately stable. There were no differences between groups (i.e., the order between watches). Sleep duration during the 06:00-12:00 h off-duty period (3 h 29 min) was significantly longer than during the 12:00-18:00 h period (1 h 47 min) and the 18:00-24:00 h period (2 h 7 min). Sleep during the 00:00-06:00 h period (4 h 23 min) was longer than all sleep periods except the 06:00-12:00 h period. There were no differences between groups. In spite of sufficient opportunities for sleep, sleep was on the average around 1-1 h 30 min shorter than the 7-7 h 30 min that is considered “normal” during a 24 h period. This is probably a consequence of the difficulty to sleep during daytime due to the alerting effects of the circadian rhythm. Also, sleepiness during the night and early mornings reached high levels, which may be explained by a combination of working close to or during the circadian trough of alertness and the relatively short sleep periods obtained. An initial suppression of sleepiness was observed during all watches, except for the 06:00-12:00 h watch. This suppression may be explained by the “masking effect” exerted by the relative high levels of activity required when taking over the responsibility of the ship. Toward the end of watches, the levels of sleepiness progressively increased to relatively high levels, at least during the 00:00-06:00 h watch. Presumably, initially high levels of activity are replaced by routine and even boredom.     

Twenty-four-hour and annual variation in onset of epistaxis in Osler disease

Osler disease is an autosomal dominant disorder of the fibrovascular tissue characterized by arteriovenous malformations with multi-systemic haemorrhages. Recurrent epistaxis is the predominant symptom in more than 90% of patients. Recent studies showed circadian and seasonal patterns in the onset of nosebleeds, similar to acute cardiovascular events, such as myocardial infarction and stroke. The aim of this study was to determine whether such patterns would also apply to the onset of epistaxis in patients with Osler disease. In all, 110 patients with Osler disease who were under treatment for recurrent epistaxis at the University Hospital of Mannheim were requested to complete a questionnaire addressing the intensity and frequency of epistaxis according to the classification of Bergler et al., as well as circadian and circannual rhythmicity in the occurrence of epistaxis according to visual analogue scales (VAS). More than half of the patients claimed to experience daily to weekly episodes of recurrent epistaxis. The occurrence of epistaxis showed a biphasic 24 h pattern, with a primary peak in the morning (05:00-8:00 h) and smaller secondary peaks in the evening (17:00-20:00 h and 21:00-00:00 h). No significant seasonal variation was found in the onset of epistaxis. However, a slight tendency, with a peak in winter months, was observed. Similar to other chronobiological studies on nosebleeds, this study showed that the 24 h pattern and seasonal tendency in the onset of epistaxis even applied to patients with Osler disease. Further investigations are necessary to determine the pathological mechanism underlying this phenomenon.     

Does circadian and seasonal variation in occurrence of acute aortic dissection influence in-hospital outcomes?

The risk of acute aortic dissection (AAD) exhibits chronobiological variations with peak onset in the morning and in winter. However, it is not known whether the time of day or season of the year of the AAD affects clinical outcomes. We studied 1,032 patients enrolled in the International Registry of Acute Aortic Dissection from January 1997 to December 2001. For circadian and seasonal analysis, the time and date of symptom onset were available for 741 and 1,007 patients, respectively, and were grouped into four 6 h periods (morning, afternoon, evening, and night) and four seasons (winter, spring, summer, and autumn). The χ² test for goodness of fit was used to evaluate non-uniformity of the time of day and time of year for critical in-hospital clinical events, including death. While highest incidence of AAD occurred in the morning and winter, clinical events (including mortality) were similar during the four different periods of the 24 h (χ²=1.9, p=0.60) and seasonal (χ²=1.2, p=0.75) periods.     

Disturbance of circadian rhythms in analgosedated intensive care unit patients with and without craniocerebral injury

Melatonin, cortisol, heart rate, blood pressure, spontaneous motor activity, and body temperature follow stable circadian rhythms in healthy individuals. These circadian rhythms may be influenced or impaired by the loss of external zeitgebers during analgosedation, critical illness, continuous therapeutic intervention in the intensive care unit (ICU), and cerebral injury. This prospective, observational, clinical study examined 24 critically ill analgo-sedated patients, 13 patients following surgery, trauma, or acute respiratory distress (ICU), and 11 patients with acute severe brain injury following trauma or cerebral hemorrhage (CCI). Blood samples for the determination of melatonin and cortisol were obtained from each patient at 2 h intervals for 24 h beginning at 18:00 h on day 1 and ending 16:00 h on day 2. Blood pressure, heart rate, body temperature, and spontaneous motor activity were monitored continuously. Level of sedation was assessed using the Ramsey Sedation Scale. The severity of illness was assessed using the APACHE-II-score. The time series data were analyzed by rhythm analysis with the Chronos-Fit program, using partial Fourier series with up to six harmonics. The 24 h profiles of all parameters from both groups of patients were greatly disturbed/abolished compared to the well-known rhythmic 24 h patterns in healthy controls. These rhythm disturbances were more pronounced in patients with brain injury. The results of this study provide evidence for a pronounced disturbance of the physiological temporal organization in ICU patients. The relative contribution of analgosedation and/or brain injury, however, is a point of future investigation.     

12-hour phase-shift of mice kidney rhodanese (thiosulfate sulfurtransferase) activity in the first two months of life

This study aimed to investigate and compare variation of renal rhodanese activity at 2^nd, 4^th and 8^th weeks of post-natal development (PND) in mice. The enzyme activity increased with age and was higher in females compared to males in all studied groups. Cosinor analysis revealed significant circadian rhythms (with period τ = 24 h) of enzyme activity in both genders with peak time shift during the PND. At the 2^nd week of PND (pre-weaning time), the circadian rhythm peaked at the beginning of light span, more precisely ≅1 HALO (Hours After Light Onset). A week after weaning (4^th week of PND), the peak time was located at the second half of photophase (≅9 HALO) in both genders. Four to six weeks later, about the 8^th week of PND, the circadian peak time was then recorded at ≅13 HALO. These findings suggest that rhodanese level and rhythm stabilization were age-dependent. Moreover, gender-related differences may stimulate discussions on the relationship between renal rhodanese and cyanide sensitivity.     

24-hour, weekly, and annual patterns in traumatic and non-traumatic surgical pediatric emergencies

24 h patterns with high frequency components in the incidence of pediatric trauma were validated and quantified in one of our earlier studies. Herein, we further explored the temporal—high frequency, 24 h, weekly (7 d), hemi-weekly (3.5 d), and annual - patterns in traumatic (1990-1997; n=15,110 events) and non-traumatic pediatric surgical emergencies (PSE) (1992-2001; n=5,593 events) as well as automobile accidents (AA) (1990-1997; n=67,712) in the County of Vaud, Switzerland. The latter served as a reference system of human adult activity and risk. Two-way ANOVA, χ2, correlation, and cosinor analyses were used as statistical tools. A 24 h pattern, reproducible from year to year, was validated in traumatic and non-traumatic PSE and AA. The 24 h patterns were not correlated and differed from one another in terms of their acrophase (peak time) and amplitude. A gender-related difference was found only in the non-traumatic time series for weekly (7 d) and hemi-weekly (3.5 d) patterns. The latter were detected in boys but not girls. No statistically significant difference was found in the acrophase and amplitude between boys and girls in the temporal patterns of other periods. An annual pattern was validated in automobile accidents (acrophase: 4th of September ±37 d (SD)) and pediatric trauma (acrophase: 14th of June ±10 d), but not in non-traumatic PSE. These results suggest that environmental modulations differ between the incidence of traumatic and non-traumatic PSE. Presumably, the two phenomena involve different aspects of the temporal organization and/or different levels of susceptibility of a set of biological rhythms to environmental factors.     

The photoperiodic entrainment and induction of the circadian clock regulating seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala)

The properties of the circadian photoperiodic oscillator have been investigated in detail only in the Japanese quail. While the study of the quail is clearly very important, one cannot simply assume that other species, especially passerines that seem to have a different circadian organization than quail, function the same way. The current set of experiments was conducted to understand the entrainment and photoinduction of the circadian photoperiodic oscillator in a passerine species, the blackheaded bunting (Emberiza melanocephala). The experimental paradigm used skeleton photoperiods with two light periods, the first called the “entraining light pulse” (E-pulse) and the second called the “inducing light pulse” (I-pulse). Three experiments were performed on photosensitive male birds (N=6-8/group). Experiment 1 investigated the effects of the temporal relationship between E- and I-pulses on photoperiodic induction. Buntings entrained to 8h:16h L:D for 4 wk were released into constant dim light (LL_dim, ∼1 lux). Beginning on subjective day 8, they received for 8 wk, E- and I-pulses only at alternate cycles. While I-pulse was 1 h and always began at zt 11.5, E-pulse varied in duration and timing (the 1h E-pulse beginning either at zt 0, zt 5, or zt 9, the 4h one beginning at zt 0 or zt 6, and the 10h one at zt 0; zeitgeber time 0=time of lights-on under 8h:16h L:D prior to release into LL_dim). A photoperiodic response was induced only when the E-pulse began at zt 0, and thus the beginning of E- and I-pulses were separated by 11.5 h. Experiment 2 determined whether the duration of the E-pulse influences the position of the photoinducible phase (φi) of the circadian photoperiodic oscillator. Birds were entrained to 1h:23h L:D or 10h:14h L:D for 2 wk, and then exposed to 1h I-pulse at zt 11.5, zt 15, or zt 18.5 for another 8 wk. Photoperiodic induction occurred at all 3 zts in birds entrained to 10 h but only at zt 11.5 in birds entrained to 1 h, which infers the circadian rhythm of photoinducibility (CRP) in buntings was re-entrained when I-pulse fell at zt 15 and after. The last experiment examined the possibility of the re-entrainment of the CRP to light pulses falling at zt 15 and after. Birds received 1h I-pulse for 8 wk at zt 15 following 2 wk of 2.5h:21.5h L:D or 3.5h:20.5h L:D, or at zt 21.5 or zt 22.5 following 2 wk of 10h:14h LD. Photoperiodic induction was consistent with the hypothesis of the re-entrainment of the CRP under these light-dark cycles. The I-pulse appeared to be interpreted as a “new dawn”, and so the photoperiodic induction was determined by the coincidence of φi with the E-pulse. These results suggest a phase-dependent action of light on the circadian oscillator regulating photoperiodic responses in the blackheaded bunting. This could be a useful strategy for a photoperiodic species to regulate its seasonal responses in nature.     

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

Entrainment of eclosion rhythm in Drosophila melanogaster populations reared for more than 700 generations in constant light environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light-dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free-running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights-on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Circadian rhythm in plasma aldosterone concentration and its seasonal modulation in the camel (Camelus dromedarius) living in the Algerian Sahara desert

Five male camels dwelling in the Algerian Sahara were studied for circadian rhythmicity in plasma aldosterone concentration and its seasonal modulation. Blood was sampled at a frequency of 1 h or less for a span of 27 h during each season of the year. The mean plasma aldosterone concentration exhibited a significant circadian rhythmicity in every season of the year. Plasma aldosterone concentration was lowest in the morning, increased in the afternoon, and generally highest in the late evening. The peak of the circadian rhythm exhibited seasonal variation; it occurred at 20:04h in October, 16:41h in December, 20:40h in March, and 24:16h in June. The rhythm's 24h mean also exhibited seasonal variability, being significantly higher in March and June compared to October.     

Twenty-four-hour pattern of angina pectoris, acute myocardial infarction and sudden cardiac death: Role of blood pressure, heart rate and rate-pressure product circadian rhythms

Population-based epidemiology and clinical case studies document a prominent 24-hour pattern in the occurrence of silent and non-silent angina pectoris (AP), acute myocardial infarction (AMI), and sudden cardiac death (SCD). When the data are summarized per 3 - 6 hour intervals of the 24 hours, the temporal pattern of these ischemic heart disease (IHD) events shows a single morning peak between 06:00 and 12:00 h in incidence. However, when the occurrence of such events is examined according to the hour of their occurrence, several studies reveal a second late-afternoon/early-evening minor peak. The true day - night pattern in AP, AMI, and SCD is unknown because the data represent nothing more than the recorded “time of day” of the events. It has been postulated that the day - night pattern in IHD events is at least in part dependent on endogenous circadian rhythms, which are synchronized by the daily routine of sleep in darkness/activity in light. Approximately 20% of the working population is involved in night and rotating shift employment; thus, “time of day” studies are not likely to accurately represent the actual “chronorisk” of vulnerable individuals to IHD events. Moreover, it is likely that the events in the persons comprising the population and clinical case studies were influenced by ongoing treatment with antihypertensive, anticoagulant, and antianginal medications. Details regarding the class, dose, and schedule of such medications are rarely if ever reported in accounts of IHD events. Many of the investigations were conducted decades ago, when short-acting antihypertensive and cardiovascular medications required twice or thrice-a-day dosing, and thus the observed day - night variations could be significantly affected by such multiple treatment timings each day. Thus, the magnitude and nature (single versus multiple peaks) of the reported day - night patterns in AP, AMI, and SCD are suspect, as are their geneses. Presently, it is hypothesized that multiple cyclic exogenous triggers (e.g. posture, physical exertion, emotional stress, and medication scheduling) superimposed upon an endogenous 24-hour susceptibility-resistance pattern that arises from circadian rhythms in heart rate, blood pressure, rate-pressure product, and haemostasis, are major contributory factors.     

Circadian response of annual weeds to glyphosate and glufosinate

Five field experiments were conducted in 1998 and 1999 in Minnesota to examine the influence of time of day efficacy of glyphosate [N-(phosphonomethyl)glycine] and glufosinate [2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid] applications on the control of annual weeds. Each experiment was designed to be a randomized complete block with four replications using plot sizes of 3×9 m. Glyphosate and glufosinate were applied at rates of 0.421 kg ae/ha and 0.292 kg ai/ha, respectively, with and without an additional adjuvant that consisted of 20% nonionic surfactant and 80% ammonium sulfate. All treatments were applied with water at 94 L/ha. Times of day for the application of herbicide were 06:00h, 09:00h, 12:00h, 15:00h, 18:00h, 21:00h, and 24:00h. Efficacy was evaluated 14 d after application by visual ratings. At 14 d, a circadian response to each herbicide was found, with greatest annual weed control observed with an application occurring between 09:00h and 18:00h and significantly less weed control observed with an application at 06:00h, 21:00h, or 24:00h. The addition of an adjuvant to both herbicides increased overall efficacy, but did not overcome the rhythmic time of day effect. Results of the multiple regression analysis showed that after environmental temperature, time of day was the second most important predictor of percent weed kill. Thus, circadian timing of herbicide application significantly influenced weed control with both glyphosate and glufosinate.