A comparison of some different methods for purifying core temperature data from humans

Nine healthy females were studied about the time of the spring equinox while living in student accommodations and aware of the passage of solar time. After 7 control days, during which a conventional lifestyle was lived under a 24h “constant routine,” the subjects lived 17 × 27h “days” (9h sleep in the dark and 18h wake using domestic lighting, if required). Throughout the experiment, recordings of wrist activity and rectal (core) temperature were taken. The raw temperature data were assessed for phase and amplitude by cosinor analysis and another method, “crossover times,” which does not assume that the data set is sinusoidal. Two different purification methods were used in attempts to remove the masking effects of sleep and activity from the core temperature record and so to measure more closely the endogenous component of this rhythm; these two methods were “purification by categories” and “purification by intercepts.” The former method assumes that the endogenous component is a sinusoid, and that the masking effects can be estimated by putting activity into a number of bands or categories. The latter method assumes that a temperature that would correspond to complete inactivity can be estimated from measured temperatures by linear regression of these on activity and extrapolation to a temperature at zero activity. Three indices were calculated to assess the extent to which exogenous effects had been removed from the temperature data by these purification methods. These indices were the daily variation of phase about its median value; the ratio of this variation to the daily deviation of phase about midactivity; and the relationship between amplitude and the square of the deviation of phase from midactivity. In all cases, the index would decrease in size as the contribution of the exogenous component to a data set fell. The purification by categories approach was successful in proportion to the number of activity categories that was used, and as few as four categories produced a data set with significantly less masking than raw data. The method purification by intercepts was less successful unless the raw data had been “corrected” to reflect the direct effects of sleep that were independent of activity (a method to achieve this being produced). Use of this purification method with the corrected data then gave results that showed least exogenous influences. Both this method and the purification by categories method with 16 categories of activity gave evidence that the exogenous component no longer made a significant contribution to the purified data set. The results were not significantly influenced by assessing amplitude and phase of the circadian rhythm from crossover times rather than cosinor analysis. The relative merits of the different methods, as well as of other published methods, are compared briefly; it is concluded that several purification methods, of differing degrees of sophistication and ease of application to raw data, are of value in field studies and other circumstances in which constant routines are not possible or are ethically undesirable. It is also concluded that such methods are often somewhat limited insofar as they are based on pragmatic or biological, rather than mathematical, considerations, and so it is desirable to attempt to develop models based equally on mathematics and biology. (Chronobiology International, 17(4), 539-566, 2000)     

The Difference Between Activity When in Bed and Out of Bed. II. Subjects on 27-Hour “Days”

Nine healthy subjects have been studied while exposed to the normal alternation of light and dark, but with their sleep and activity pattern adjusted to a 27-h “day” for 17 imposed “days.” Rectal temperature showed clearly the competing influences of 27-h and 24-h components, and these were separated by the method of “purification.” The method indicated that the endogenous component had a constant amplitude throughout the experiment and remained entrained to solar (24-h) time; by contrast, the exogenous component followed the imposed 27-h “day” and increased rectal temperature in proportion to the amount of subjects' activity. Wrist movement was used to assess activity while in bed (attempting sleep) and out of bed (when naps were forbidden). While these results confirmed adherence of the subjects to the imposed 27-h “days,” they also showed that the dichotomy between “out of bed” activity and “in bed” inactivity depended on the phase relationship between endogenous (24h) and exogenous (27h) components. Thus, the dichotomy was highest and was equal to that during control days (with a conventional 24-h life-style) when the two components were in phase and lowest when the solar and imposed day were in antiphase. This was due to changes in activity, both during time spent in bed and out of bed.

We confirm that this protocol can produce valuable information about the properties of the circadian system in humans and the value of the process of purification of temperature data. We have established also that the very simple and noninvasive measurement of wrist movement, coupled with its use to calculate dichotomy indices, provides valuable information that both confirms and extends the results obtained from the more conventional (butalso more invasive) measurement of rectal temperature.     

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)     

Lack of evidence that feedback from lifestyle alters the amplitude of the circadian pacemaker in humans

Two groups of healthy subjects were studied indoors, first while living normally for 8 days (control section) and then for 18 × 27h “days” (experimental section). This schedule forces the endogenous (body clock-driven) and exogenous (lifestyle-driven) components of circadian rhythms to run independently. Rectal temperature and wrist movement were measured throughout and used as markers of the amplitude of the circadian rhythm, with the rectal temperature also “purified” by means of the activity record to give information about the endogenous oscillator. Results showed that, during the experimental days, there were changes in the amplitude of the overt temperature rhythm and in the relative amounts of out-of-bed and in-bed activity, both of which indicated an interaction between endogenous and exogenous components of the rhythm. However, the amplitude and the amount of overlap were not significantly different on the control days (when endogenous and exogenous components remained synchronized) and those experimental days when endogenous and exogenous components were only transiently synchronized; also, the amplitudes of purified temperature rhythms did not change significantly during the experimental days in spite of changes in the relationship between the endogenous and exogenous components. Neither result offers support for the view that the exogenous rhythm alters the amplitude of oscillation of the endogenous circadian oscillator in humans.     

The development of new purification methods to assess the circadian rhythm of body temperature in Mongolian gerbils

Six Mongolian gerbils were studied for 8-10d while housed in separate cages in a 12:12h light-dark (L-D) cycle (lights on at 07:00h). Recordings of body temperature, heart rate, and spontaneous activity were made throughout. The temperature and heart rate rhythms were “purified” to take into account the effects of activity, and then the rhythm of temperature was further purified to take into account other masking influences (“non-activity masking effects” or NAME,). The methods employed in the purification processes involved linear regression analysis or analysis of covariance, the latter using functions of activity and NAME as covariates. From these methods, it was possible to obtain not only an estimate of the endogenous component of the temperature rhythm but also a measure of circadian changes in the sensitivity of temperature to masking effects.

Even though all purification methods removed many of the effects of spontaneous activity from the temperature record, there remained temperature fluctuations at the L-D and D-L transitions that appeared to be independent of activity. The NAME was of only very marginal value in the purification process. Comparison of the purification methods indicated that the linear methods were inferior (both from a biological viewpoint and when the results were compared mathematically) to those that allowed the rate of rise of temperature due to increasing amounts of activity to become progressively less. The sensitivity of temperature and heart rate to the masking effects of activity showed a circadian rhythm, with sensitivities in the resting phase being greater than those in the active phase. These findings are compatible with the view that thermoregulatory reflexes are induced by spontaneous activity of sufficient amount, and that there is a circadian rhythm in the body temperature at which these reflexes are initiated and in their effectiveness.     

Effects of Nocturnal Shiftwork on Mood States of Student Nurses

Daily mood changes were monitored over successive 24-h periods using the Profile of Mood States (POMS) (3) to assess the effect of nocturnal shiftwork on mood. Twenty-three student nurses, age range 19-24 years, were studied throughout their first experience of nocturnal shiftwork. The POMS was administered over four complete solar days during a 12-week period that included an 8-week block of night work. Five POMS dimensions displayed circadian rhythmicity. vigor-activity; fatigue-inertia; confusion-bewilderment; friendliness; and total-mood-disturbance. These five dimensions were sensitive to changes in living patterns, showing phase shifts in their circadian rhythms when subjects alternated between diurnal and nocturnal living patterns. The dimensions were also observed to be sensitive to adjustment to two different nocturnal shiftwork schedules. The subjects who worked “four on, three off showed similar phase shifts to the subjects who worked “eight on, seven off,” suggesting that mood adjustment takes place by the fourth night of a rotation of nights. The “commitment” of the students to the nocturnal living pattern was thought to have a bearing on the adaptation of the students to the nocturnal shifts, as regards mood.     

Temperature profiles, and the effect of sleep on them, in relation to morningness-eveningness in healthy female subjects

There were 15 healthy female subjects, differing in their position on the “morningness-eveningness” scale, studied for 7 consecutive days, first while living a sedentary lifestyle and sleeping between midnight and 08:00 and then while undergoing a “constant routine.” Rectal temperature was measured at regular intervals throughout this time, and the results were subjected to cosinor analysis both before and after “purification” for the effects of physical activity. Results showed that there was a phase difference in the circadian rhythm of core temperature that was associated with the morningness score, with calculations that “morning types” would be phased earlier than “evening types” by up to about 3h. This difference in phase (which was also statistically significant when the group was divided by a median split into a “morning group” and an “evening group”) could not be attributed to effects of waking activity and existed in spite of the subjects keeping the same sleep-wake schedule. Moreover, it persisted when the subjects' data had been purified and when the data were obtained from the constant routine. That is, there was an endogenous component to this difference in phase of the core temperature. The morning group also showed a greater fall of core temperature during sleep; this was assessed in two ways, the main one being a comparison of constant routine and nychthemeral data sets after correction for any effects of activity. Even though the morning group was sleeping at a later phase of their circadian temperature rhythm than was the evening group, neither group showed a fall of temperature due to sleep that varied with time elapsed since the temperature acrophase. It is concluded that another factor that differs between morning and evening types is responsible for this difference. (Chronobiology International, 18(2), 227-247, 2001)     

Regression Models for the Estimation of Orcadian Rhythms in the Presence of Effects Due to Masking

The estimation of human circadian rhythms from experimental data is complicated by the presence of “masking” effects associated with the sleep-wake cycle. The observed rhythm may include a component due to masking, as well as the endogenous component linked to a circadian pacemaker. In situations where the relationship between the sleep-wake cycle and the circadian rhythm is not constant, it may be possible to obtain individual estimates of these two components, but methods commonly used for the estimation of circadian rhythms, such as the cosinor analysis, spectral analysis, average waveforms and complex demodulation, have not generally been adapted to identify the modulations that arise from masking. The estimates relate to the observed rhythms, and the amplitudes and acrophases do not necessarily refer to the endogenous rhythm.

In this paper methods are discussed for the separation of circadian and masking effects using regression models that incorporate a sinusoidal circadian variation together with functions of time since sleep and time during sleep. The basic model can be extended to include a time-varying circadian rhythm and estimates are available for the amplitude and phase at a given time, together with their joint confidence intervals and tests for changes in amplitude and acrophase between any two selected times. Modifications of these procedures are discussed to allow for non-sinusoidal circadian rhythms, non-additivity of the circadian and time-since-sleep effects and the breakdown of the usual assumptions concerning the residual errors.

This approach enables systematic masking effects associated with the sleep-wake cycle to be separated from the circadian rhythm, and it has applications to the analysis of data from experiments where the sleep-wake cycle is not synchronized with the circadian rhythm, for example after time-zone transitions or during irregular schedules of work and rest.     

Some factors influencing the sensitivity of body temperature to activity in neonates

In adult humans, core temperature is influenced by activity; the sensitivity of core temperature to such effects shows a phase dependence and is also influenced by the environment and whether the individual is asleep or awake. We have investigated if similar effects are evident in neonates, in whom thermoregulation and the circadian rhythm of core temperature are not fully developed. Eleven full-term, healthy babies were studied singly (light 07:00-19:00) at 2 days of age and again 4 weeks after birth; between these times, they were tended routinely on a communal ward. On study days, 10-minute recordings were made of rectal and skin (abdominal) temperature, heart rate (HR), and behavioral state. Sensitivities of the temperatures to activity (“arousal”) were assessed throughout the 24h by measuring the gradient of (temperature/HR). Sensitivities measured at 01:00, 05:00, 09:00, 13:00,17:00, and 21:00 were used as dependent variables in stepwise regression and linear regression analyses, with “subjects” “light versus dark”, “behavioral state”, and “difference between time of measurement and the acrophase of the endogenous component of the temperature rhythm” (ignoring sign) as possible predictors. (Acrophases of the temperature rhythms had been estimated from 24h data purified using the behavioral state record.) Light versus dark acted as a significant predictor of the sensitivity of rectal temperature to arousal on day 2 and week 4, the sensitivity increasing in the light, and there was limited evidence for behavioral state acting as a predictor on day 2. Neither factor was a significant predictor when the sensitivity of the babies' skin temperatures to arousal was investigated. There was also some evidence that the difference between the time of measurement and the temperature acrophase acted as a predictor of sensitivity to arousal in both rectal (day 2) and skin (week 4) temperature, with larger differences decreasing the sensitivity. These results indicate that there are masking effects on body temperature due to arousal in neonates, the size of which depends on both internal and external factors. However, this sensitivity of temperature to arousal shows differences from the sensitivity of temperature to physical activity in both adult humans and adult mice. One possible explanation of this result is that temperature regulation and the circadian system are not fully developed in humans at this age. (Chronobiology International, 17(5), 679-692, 2000)     

Sleep and Circadian Rhythms of Temperature and Urinary Excretion on a 22.8 hr “Day”

Two groups of subjects (total N = 6) were studied in an isolation chamber for a period of 3 weeks whilst living on a 22.8 hr “day”. Regular samples of urine were taken when the subjects were awake, deep body temperature was recorded continuously and polygraphic EEG recordings were made of alternate sleeps. The excretion in the urine of potassium, sodium, phosphate, calcium and a metabolite of melatonin were estimated.

Measurements of the quantity and quality of sleep were made together with assessments of the temperature profiles associated with sleep. In addition, cosinor analysis of circadian rhythmicity in urinary variables and temperature was performed.

The 22.8 hr “days” affected variables and subjects differently. These differences were interpreted as indicating that the endogenous component of half the subjects adjusted to the 22.8 hr “days” but that, for the other three, adjustment did not occur. When the behaviour of different variables was considered then some (including urinary potassium and melatonin, sleep length and REM sleep) appeared to possess a larger endogenous component than others (for example, urinary sodium, phosphate and calcium), with rectal temperature behaving in an intermediate manner. In addition, a comparison between different rhythms in any subject enabled inferences to be drawn regarding any links (or lack of them) that might exist between the rhythms. In this respect also, there was a considerable range in the results and no links between any of the rhythms appeared to exist in the group of subjects as a whole.

Two further groups (total N=8) were treated similarly except that the chamber clock ran at the correct rate. In these subjects, circadian rhythms of urinary excretion and deep body temperature (sleep stages and urinary melatonin were not measured) gave no evidence for deterioration. We conclude, therefore, that the results on the 22.8 hr “day” were directly due to the abnormal “day” length rather than to a prolonged stay in the isolation chamber.     

"Live memory" of the cell, the other hereditary memory of living systems

We propose to designate by the term “live memory” of the cell, the cytoplasmic memory. This phenomenon consists of non-genetic memory, but nevertheless includes transmission function, which may be “hereditary” via the ovum, from mother cell to daughter cell, or simply within the same cell from instant t to instant t + 1. To understand this notion of “live memory”, its role and interactions with DNA must be resituated; indeed, operational information belongs as much to the cell body and to its cytoplasmic regulatory protein components and other endogenous or exogenous ligands as it does to the DNA database. We will see in Section 2, using examples from recent experiments in biology, the principal roles of “live memory” in relation to the four aspects of cellular identity, memory of form, hereditary transmission and also working memory.     

The Effect of Activity on the Waking Temperature Rhythm in Humans

Nine healthy female subjects were studied when exposed to the natural light-dark cycle, but living for 17 “days” on a 27h day (9h sleep, 18h wake). Since the circadian endogenous oscillator cannot entrain to this imposed period, forced desynchronization between the sleep/activity cycle and the endogenous circadian temperature rhythm took place. This enabled the effects of activity on core temperature to be assessed at different endogenous circadian phases and at different stages of the sleep/activity cycle. Rectal temperature was measured at 6-minute intervals, and the activity of the nondominant wrist was summed at 1-minute intervals. Each waking span was divided into overlapping 3h sections, and each section was submitted to linear regression analysis between the rectal temperatures and the total activity in the previous 30 minutes. From this analysis were obtained the gradient (of the change in rectal temperature produced by a unit change in activity) and the intercept (the rectal temperature predicted when activity was zero). The gradients were subjected to a two-factor analysis of variance (ANOVA) (circadian phase/ time awake). There was no significant effect of time awake, but circadian phase was highly significant statistically. Post hoc tests (Newman-Keuls) indicated that gradients around the temperature peak were significantly less than those around its trough. The intercepts formed a sinusoid that, for the group, showed a mesor (±SE) of 36.97 (±0.12) and amplitude (95% confidence interval) of 0.22°C (0.12°C, 0.32°C). We conclude that this is a further method for removing masking effects from circadian temperature rhythm data in order to assess its endogenous component, a method that can be used when subjects are able to live normally. We suggest also that the decreased effect of activity on temperature when the endogenous circadian rhythm and activity are at their peak will reduce the possibility of hyperthermia.     

Induction of fever in a teleost fish (Lepomis gibbosus, L.)

Although fishes are ectotherms they are nevertheless able to thermoregulate behaviorally by selecting appropriate water temperatures (1). In a temperature gradient fish will congregate to a species-specific range of preferred temperature (“final thermal referendum”) which is unaffected by previous thermal history of the individual (2,3). Several aquatic (and terrestrial) ectothermic vertebrates have been found to exhibit “behavioral fever” which is manifested as an increase in preferred temperature above the final thermal preferendum (4). Fever can be elicited by pyrogens: whole bacteria (alive or killed), components of bacterial cellwall (endotoxins), endogenous pyrogens, prostaglandins or from several other sources (5). Since the results with fever induction in fish using whole bacteria or endotoxins are very scarce the aim of the present work was to compare possible thermoregulatory effects of endotoxins and prostaglandins in the same species (Lepomis gibbosus, L.) by means of identical methods.     

Can Insulated Skin Temperature Act as a Substitute for Rectal Temperature when Studying Circadian Rhythms?

We measured rectal, lateral chest wall, and axillary temperature every half hour for at least 24 h while subjects were living normal life-styles and keeping a sleep/activity diary. We then used a purification method to estimate the decrease of temperature due to sleep and the increases due to sitting, standing, walking, or exercising, as well as the parameters of the cosine curve that described the “purified data.” Cosinor analysis of raw and purified data showed that the acrophases from both skin sites were much more variable and up to 8 h later than were those from the rectum (particularly if exercise had been taken), even though the acrophases from the two skin sites were similar to each other. For rectal temperature, there was an increase in the size of the masking effect as activity progressed through the sequence: sitting, standing or walking, exercising. In contrast, for both chest wall and axillary temperatures, although sitting produced masking effects similar to those for rectal temperature, masking effects due to standing or walking and exercising were much smaller, and sometimes they were even less than the masking effects due to sitting. These results indicate that our measurements of cutaneous temperature did not act as a substitute for rectal temperature, particularly when the subject was physically active rather than sedentary.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

"Demasking" the temperature rhythm after simulated time zone transitions.

Simulated time zone transitions were performed in an isolation unit upon groups of one to four human subjects. In the first series of experiments, the adjustment of the circadian rhythm of body temperature, measured in the presence of sleep and other masking factors, was assessed by cosinor analysis and by cross-correlation methods. These methods modeled the circadian timing system either as a single component or as the sum of two components, those due to exogenous and endogenous influences. The one-component models described a more rapid adjustment of the temperature rhythm to the time zone transition than did the two-component models; we attribute this difference to the masking effects of the exogenous component. In a second series of experiments, we showed that the shift of the endogenous component, as assessed by the two-component models, was not significantly different from that measured during constant routines. The results also showed that, if the zeitgebers were phased in advance of the endogenous component, then advances of the endogenous component were produced only if this mismatch was less than about 10 hr. Mismatches greater than this, and cases where the zeitgebers were delayed with respect to the endogenous component, both produced delays of the endogenous component. We conclude that the two-component cross-correlation methods can be used to estimate shifts of the endogenous component of a circadian rhythm in the presence of masking factors. They are therefore an alternative to constant routines when these latter are impracticable to carry out.     

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.     

Effect of melatonin on sleep quality of COPD intensive care patients: a pilot study

Sleep deprivation is extremely common in the intensive care unit (ICU), and this lack of sleep is associated with low melatonin secretion. The objective of the current study was to explore the effect of exogenous melatonin administration on sleep quality in patients hospitalized in the pulmonary intensive care unit (ICU). We performed a double-blind, placebo-controlled study in the pulmonary ICU of a tertiary care hospital. Eight adult patients hospitalized in the pulmonary ICU with respiratory failure caused by exacerbation of chronic obstructive pulmonary disease (COPD) or with pneumonia were studied. Patients received either 3 mg of controlled-release melatonin or a placebo at 22:00, and sleep quality was evaluated by wrist actigraphy. Treatment with controlled-release melatonin dramatically improved both the duration and quality of sleep in this group of patients. Our results suggest that melatonin administration to patients in intensive care units may be indicated as a treatment for sleep induction and resynchronization of the “biologic clock.” This treatment may also help in the prevention of the “ICU syndrome” and accelerate the healing process. (Chronobiology International, 17(1), 71-76, 2000)     

Masking in Humans: The Problem and Some Attempts to Solve IT

Different types of masking are discussed together with an account of the masking effect that the sleep-wake cycle exerts upon the circadian rhythms of body temperature and urinary excretion. The relative importance to masking of the several components of differences between sleeping and wakefulness are then assessed.

Means to deal with the problem of masking fall into two major categories. These attempt to minimise masking effects by protocols such as constant routines or control days, and mathematical models which separate results obtained in the presence of masking influences into endogenous and exogenous components. (The problem of the extent to which masking influences can render the endogenous component of a rhythm an impure reflection of the internal oscillator is considered also.) These different techniques are compared with respect to their usefulness and assumptions.

Finally, a brief speculation is given of the usefulness of masking.     

Disappearance of a 32 000 D chromatin polypeptide during early erythroid differentiation of Friend leukemia cells

The patterns of non-histone chromatin proteins have been investigated in dimethyl sulphoxide (DMSO)-stimulated Friend leukemia cells (FLC) undergoing the “early” events of erythroid differentiation. Sucrose-purified whole nuclei were lysed and proteins extracted with 6 M urea and 4 M guanidium hydrochloride. The proteins were analysed in SDS-and in SDS-urea-polyacrylamide gel electrophoresis. The disappearance of a 32 000 D chromatin protein component was observed in cells of the 745A “inducible” line harvested as early as 24 h after DMSO treatment, as compared with untreated 745A cells and to cells harvested 6 and 12 h after DMSO addition to the cultures. By contrast, a 32 000 D chromatin protein component is always present in untreated as well as in DMSO-treated cells of the “uninducible” Fw line of FLC.