Circadian Rhythm of Serotonin Binding in Rat Brain—II. Influence of Sleep Deprivation and Imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P < 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed though the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak valueof 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

Circadian rhythm of serotonin binding in rat brain--II. Influence of sleep deprivation and imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P less than 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed through the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak value of 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

Memory for semantically related and unrelated declarative information: the benefit of sleep, the cost of wake

Numerous studies have examined sleep's influence on a range of hippocampus-dependent declarative memory tasks, from text learning to spatial navigation. In this study, we examined the impact of sleep, wake, and time-of-day influences on the processing of declarative information with strong semantic links (semantically related word pairs) and information requiring the formation of novel associations (unrelated word pairs). Participants encoded a set of related or unrelated word pairs at either 9 am or 9 pm, and were then tested after an interval of 30 min, 12 hr, or 24 hr. The time of day at which subjects were trained had no effect on training performance or initial memory of either word pair type. At 12 hr retest, memory overall was superior following a night of sleep compared to a day of wakefulness. However, this performance difference was a result of a pronounced deterioration in memory for unrelated word pairs across wake; there was no sleep-wake difference for related word pairs. At 24 hr retest, with all subjects having received both a full night of sleep and a full day of wakefulness, we found that memory was superior when sleep occurred shortly after learning rather than following a full day of wakefulness. Lastly, we present evidence that the rate of deterioration across wakefulness was significantly diminished when a night of sleep preceded the wake period compared to when no sleep preceded wake, suggesting that sleep served to stabilize the memories against the deleterious effects of subsequent wakefulness. Overall, our results demonstrate that 1) the impact of 12 hr of waking interference on memory retention is strongly determined by word-pair type, 2) sleep is most beneficial to memory 24 hr later if it occurs shortly after learning, and 3) sleep does in fact stabilize declarative memories, diminishing the negative impact of subsequent wakefulness.     

Diurnal Variations of Beta-Endorphin at Rest and After Moderate Intensity Exercise

To determine the effect of time of day on circulating beta-endorphin concentrations 14 men exercised at 75% of their maximal capacity at 0600, 1200, 1800 and 2400 hr. Each trial was separated by 3-5 days and preceded by a normal sleep cycle except for the 0600 hr trials which was preceded by 6 hr sleep. Resting physiological data indicated normal diurnal variations in heart rate, core temperature and oxygen uptake, being lowest during the 0600 hr trials and highest during the 1800 hr trials. Resting plasma beta-endorphin concentrations averaged 11.9 +/- 8.4 pmol/l during the 0600 hr trials, significantly greater than the 2400 hr trials (6.4 +/- 3.6 pmol/l; P less than 0.05). No other significant differences existed at rest. Post exercise beta-endorphin concentrations were elevated and found to be inversely related to time of day with the 0600 hr trials having the highest mean (25.7 +/- 14.7) and the 2400 hr trials the lowest (14.7 +/- 8.3). These data suggest that the plasma beta-endorphin concentrations at rest and after exercise are affected by the time of day. The results also suggest that the changes in beta-endorphin associated with exercise are not major contributors to cardiorespiratory control or changes in psychological effect associated with exercise.     

Daily profiles of aldosterone levels during normal, high and low sodium intake in patients with essential hypertension.

The episodic secretion of aldosterone depends on the dietary sodium intake, alterations in posture and follows ACTH circadian rhythm. Aldosterone daily profiles have been studied in 23 supine essential hypertensive patients on normal sodium intake. Secretory pulses at a frequency of two to five pulses per 12 hr have occurred, independent of PRA levels. Among 13 patients with normal PRA two lost pulsatility when sodium was loaded (10 g/24 hr) and the same happened with two others on sodium restricted diet (2 g/24 hr). These results suggest a profound effect of dietary sodium intake on the pulsatile pattern of aldosterone secretion, particularly in normal PRA essential hypertension.     

The internal circadian clock increases hunger and appetite in the evening independent of food intake and other behaviors

Objective:

Despite the extended overnight fast, paradoxically, people are typically not ravenous in the morning and breakfast is typically the smallest meal of the day. We assessed whether this paradox could be explained by an endogenous circadian influence on appetite with a morning trough, while controlling for sleep/wake and fasting/feeding effects.

Design and Methods:

Twelve healthy non‐obese adults (six males; age, 20‐42 years) were studied throughout a 13‐day laboratory protocol that balanced all behaviors, including eucaloric meals and sleep periods, evenly across the endogenous circadian cycle. Participants rated their appetite and food preferences by visual analog scales.

Results:

There was a large endogenous circadian rhythm in hunger, with the trough in the biological morning (8 AM) and peak in the biological evening (8 PM; peak‐to‐trough amplitude = 17%; P = 0.004). Similarly‐phased significant endogenous circadian rhythms were present in appetites for sweet, salty and starchy foods, fruits, meats/poultry, food overall, and for estimates of how much food participants could eat (amplitudes 14‐25%; all P < 0.05).

Conclusions:

In people who sleep at night, the intrinsic circadian evening peak in appetite may promote larger meals before the fasting period necessitated by sleep, whereas the circadian morning trough would theoretically facilitate the extended overnight fast. Furthermore, the circadian decline in hunger across the night would theoretically counteract the fasting‐induced hunger increase that could otherwise disrupt sleep.     

To sleep, to strive, or both: how best to optimize memory

While numerous studies have shown that a night of sleep profits memory relative to wake, we still have little understanding about what factors mediate this effect of sleep. A clear understanding of the dynamics of this effect of sleep beyond the initial night of sleep is also lacking. Here, we examined the effect of extrinsic rewards on sleep-dependent declarative memory processing across 12 and 24 hr training-retest intervals. Subjects were either paid based on their performance at retest ($1 for each correct answer), or received a flat fee for participation. After a 12 hr interval we observed pronounced benefits of both sleep and reward on memory. Over an extended 24 hr interval we found 1) that an initial night of sleep partially protects memories from subsequent deterioration during wake, and 2) that sleep blocks further deterioration, and may even have a restorative effect on memory, when it follows a full day of wake. Interestingly, the benefit imparted to rewarded (relative to unrewarded) stimuli was equal for sleep and wake subjects, suggesting that the sleeping brain may not differentially process rewarded information, relative to wake. However, looking at the overall impact of sleep relative to reward in this protocol, it was apparent that sleep both imparted a stronger mnemonic boost than reward, and provided a benefit to memory regardless of whether it occurred in the first or the second 12 hrs following task training.     

Polyphasic Wake/Sleep Episodes in the Fire Ant, Solenopsis Invicta

Sleep is a well-studied biological process in vertebrates, particularly birds and mammals. Less is know about sleep in solitary and social invertebrates, particularly the ants. This paper reports a study of light/dark periods on worker activity as well as sleep location, posture and the wake/sleep cycles of fire ant workers and queens located in an artificial nest chamber. Workers slept in one of three locations: on the ceiling, against the chamber wall or in the center of the chamber floor. Workers on the ceiling or against the chamber wall slept for longer periods than those at the center of the chamber floor where most grooming and feeding activity occurred. When sleeping, queens huddled together. Their close contact generated synchronized wake/sleep cycles with each other. Sleep posture was distinctly different than wake posture. During deep sleep, queens and workers folded their antennae and were non-responsive to contact by other ants. Another indicator of deep sleep was rapid antennal movement (RAM sleep). Sleep episodes were polyphasic. Queens averaged ~92 sleep episodes per day, each episode lasting ~6 min, for a total of ~9.4 h of sleep per day. Workers averaged ~253 sleep episodes lasting 1.1 min each for a total of ~4.8 h of sleep per day. Activity episodes were unaffected by light/dark periods. Workers were hypervigilant with an average of 80% of the labor force completing grooming, feeding or excavation tasks at any given time. These findings reinforce the parental exploitation hypothesis—sterile workers are a caste of disposable, short-lived helpers whose vigilance and hyperactivty increases the queen’s fitness by buffering her and her fertile offspring from environmental stresses.     

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.     

Developmental profile of intracellular compatments during Allium cepa root cell germination

The efflux kinetics of ⁸⁶Rb from 24 hr preloaded Allium cepacv. Evergreen Long White Bunching root tip cells through thefirst 120 hr of germination have been documented. Estimatesof the percent of isotope, the calculated amount present andthe halftime of efflux of the three root cell compartments presentwere made. Of the total isotope present the % in the vacuoledecreased from maxima of 64% to 40% with germination time, whilethe % of the isotope in the cytoplasm remained uniform near39%. The free space increased maximally throughout germinationto 20% by 120 hr. Rb content, based on specific activity calculations,of the slowest, intermediate and fastest compartments was seento rise from low levels at 52 hr germination toward maximalvalues by 96 hr. Speculation as to when a germinating root becomesfunctionally mature was attempted by comparing the times ofonset of major macromolecule synthesis (DNA, RNA, protein) tothat of maximal Rb content. Stable mitotic and DNA syntheticindices occurred at the 10 mm (72 hr) stage with the initiationof major macromolecules syntheses occurring prior to 2 mm (40hr). ⁸⁶Rb efflux data suggests that the compartments do notcontain maximal amounts until the 22 mm (96 hr) stage. (Received December 26, 1978; )     

Porphyrin profiles in blood and urine as a biomarker for exposure to various arsenic species.

A sensitive method using HPLC with fluorescence detection has been established for the measurement of porphyrins in biological materials. The assay recoveries were 88.0+/-1.8% for protoporphyrin IX in the blood, and ranged from 98.3+/-2.7% to 111.1+/-7.4% for various porphyrins in the urine. This method was employed to investigate the altered porphyrin profiles in rats after a single dose of various arsenicals including soluble sodium arsenate and sodium arsenite, and the relatively insoluble calcium arsenite, calcium arsenate and arsenic-contaminated soils at dose rates of 5 mg/kg or 0.5 mg/kg body weight. Porphyrin concentrations increased within 2448 hr after the arsenic treatment in blood and urine. Protoporphyrin IX is the predominant porphyrin in the blood. In rats administered 5 mg As(III)/kg body weight, protoporphyrin IX concentration elevated to 123% of the control values in rats, 24 hr after the treatment. Higher increases were recorded in the urinary protoporphyrin IX (253% at 24 hr; 397% on day 2), uroporphyrin (121% at 24 hr; 208% on day 2) and coproporphyrin III (391% at 24 hr; 304% on day 2), while there was no significant increase (109% on day 3) observed in the urinary coproporphyrin I excretion. In rats administered 5 mg As(V)/kg, urinary excretion of protoporphyrin LX, uroporphyrin, coproporphyrin III and coproporphyrin I elevated to the maximum levels by 48 hr with the corresponding percentage values compared to the control being 177%, 158%, 224% and 143%, respectively. In rats dosed with 5 mg As(III)/kg, the increases (expressed as % of the control values) of protoporphyrin IX in the blood were in the order: sodium arsenite (144%) > sodium arsenate (125%) > calcium arsenite (123%) > calcium arsenate. In contrast, there was no significant increase of protoporphyrin IX, when the six arsenic-contaminated cattle dip soils and nine copper chrome arsenate (CCA-contaminated) soils were administered to the rats. Probable explanations are discussed.     

Coiling is Not Essential to Anhydrobiosis by Aphelenchus avenae on Agar Amended with Sucrose

The roles of preconditioning and coiling upon entrance into anhydrobiosis by Aphelenchus avenae were tested via video-assisted analysis at 25²°C. Fourth-stage juveniles or young adults of A. avenae were individually placed on 5% agar containing 0.8 M sucrose. Nematodes became quiescent within 3 hr, then gradually resumed a low level of activity and assumed a coiled posture. High desiccation survival rate was recorded when nematodes were incubated on agar for more than 6 hr; the survival rates were 0%, 3%, 73%, and 92% for 0, 2, 6, and 12 hr on agar, respectively. All nematodes placed on agar for 24 hr or more revived after rehydration following desiccation. Once nematodes were on agar for a sufficient time, no difference in desiccation survival was observed between nematodes taking a coiled posture and those uncoiled artificially. Based on these results, exposure to osmotic stress for 6 hr can prepare A. aveae physiologically for anhydrobiosis, but coiling does not appear to be a physiological requirement for desiccation in survival.     

Combined serial ultrasonography and fecal progestin analysis for reproductive evaluation of the female white rhinoceros (Ceratotherium simum simum): Preliminary results

Ultrasonographic examinations of one multiparous 33-year-old female southern white rhinoceros (Ceratotherium simum simum) resulted in documentation of the animal's estrous cycle, elucidation of the timing of ovulation in relation to estrus, and ultrasonographic evidence of endometritis and associated early embryonic death. The preovulatory follicle was observed to change in shape from spherical to pear-shape (n = 3) and to reach a mean follicular diameter of ˜30 mm (n = 4) in the 48 hr preceding estrus. An ovulation site in the location of the preovulatory follicle was observed to occur within 24 hr postbreeding on one occasion. Both pregnancies monitored in this female in 1995 were lost by day 28 postovulation, with collapse of the embryonic vesicle documented via ultrasound. Ultrasonographic evidence of endometritis was observed in this female and was characterized by small quantities of anechoic intrauterine fluid collections (5–20 mm diameter) in late diestrus (n = 4, mean day observed was 20.5 days postovulation, with a range of 18–24 days). Fecal samples collected at the time of ultrasound were evaluated via radioimmunoassay for progesterone metabolites. A substantial rise in fecal progestins was not identified until 7–9 days postovulation. This study illustrates the value of combining the complementary techniques of ultrasonographic “mapping” of events with fecal hormone assay to enhance the accuracy of reproductive monitoring. Zoo Biol 16:445–456, 1997. © 1997 Wiley-Liss, Inc.     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.     

Effects of textile dye stuff effluent on food utilization of Cyprinus carpio (Linn.)

Dye stuff effluent at 15% caused 100% mortality within 24 hr; 96 hr LC50 value being 10% concentration. Prolonged exposure of C. carpio to chosen sublethal concentrations (1, 3, 5, 7 or 9%) reduced the food intake and increased the maintenance cost. Exposure also caused significant reduction in growth rate and conversion efficiency; these values averaged to 11 mg/g/day and 28% in the control group (0% concentration) respectively and decreased to 0.8 mg/g/day and 4% respectively at 9% concentration.     

Trends in stature in the South Australian Aboriginal Murraylands

Millennial and secular changes in body height of prehistoric and recent Aboriginal South Australians are investigated. Skeletal remains of 55 male and 40 female individuals who were excavated at Roonka on the River Murray were dated from 9800 to 100 years BP. Stature was reconstructed by using humerus, femur, and tibia ratios to stature derived from Abbie's (1975) data on living Aborigines and the Trotter-Gleser method for blacks. The respective averages were 1,652 mm and 1,665 mm for males and 1,527 mm and 1,549 mm for females. In 1996/1997, statures of 27 adult males and 21 adult females were measured in Aboriginal centers of Gerard and Raukkan (Point McLeay) on the Lower River Murray. These people, as far as it can be ascertained, are the descendants of the people from Roonka. Their statures were adjusted for the stature loss with age, so that the data represent young individuals (≤30 years of age). The average male stature was 1,712 mm, and the average female stature was 1,567 mm. Data collected by Wood Jones and Campbell in 1924 for Aboriginal South Australians show that young adult male stature was 1,668 mm (n = 6), and female stature was 1,552 mm (n = 4). Slopes of regressions of individual statures on radiocarbon dates and on dates of birth are not significantly different from zero. The same is true for regressions of individual long bone lengths on radiocarbon dates. It can be concluded that there was little change in stature of Aboriginal South Australians from prehistoric to recent times. Regressions of individual age-corrected heights on birth dates (1860–1980) of Aboriginal men and women measured in 1924 and in 1996 further indicate no significant increase in height in either sex. Am J Phys Anthropol 106:505–514, 1998. © 1998 Wiley-Liss, Inc.     

Entrainment of A Free-Running Human with Bright Light?

The case of a 40-year-old sighted woman with free-running sleep-wake and melatonin rhythms is presented. The subject was studied for 102 days. During the pre-treatment period, both the sleep-wake and melatonin rhythms had a period of 25.1 hr, similar to the average period of humans living in temporal isolation. Treatment consisted of bright artifical light exposure (2500 lx Vita-Lite) for 2 hr each day upon awakening. Clock time of light exposure was held constant for 6 days and then slowly advanced until the subject was arising at her desired time of day. The subject continued the light treatment at home and was able to live on a 24-hr day for the 30-day follow-up study. While other factors may be operating in this situation, it is possible that the light treatment caused the stabilization of the free-running rhythms, advancement to a normal phase and entrainment to the 24-hr day. We suspect that the tendency to free-run was related to sleep onsets that were abnormally delayed relative to the circadian phase response curve for light. By scheduling a 2-hr pulse of bright light each morning, this tendency to delay would be counteracted by light-induced advances, resulting in normal entrainment.     

Circannual Variation of Cell Proliferation in Lymphoid Organs and Bone Marrow of Dbf1 Male Mice on Three Lighting Regimens

The case of a 40-year-old sighted woman with free-running sleep-wake and melatonin rhythms is presented. The subject was studied for 102 days. During the pre-treatment period, both the sleep-wake and melatonin rhythms had a period of 25.1 hr, similar to the average period of humans living in temporal isolation. Treatment consisted of bright artifical light exposure (2500 lx Vita-Lite) for 2 hr each day upon awakening. Clock time of light exposure was held constant for 6 days and then slowly advanced until the subject was arising at her desired time of day. The subject continued the light treatment at home and was able to live on a 24-hr day for the 30-day follow-up study. While other factors may be operating in this situation, it is possible that the light treatment caused the stabilization of the free-running rhythms, advancement to a normal phase and entrainment to the 24-hr day. We suspect that the tendency to free-run was related to sleep onsets that were abnormally delayed relative to the circadian phase response curve for light. By scheduling a 2-hr pulse of bright light each morning, this tendency to delay would be counteracted by light-induced advances, resulting in normal entrainment.     

Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

When plasma from third instar larvae of the fleshfly, Sarcophaga barbarta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 65% to ammonium sulphate, and it was found that the 50% fraction contained 95% of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration.The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7 day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.o.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme.The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 50% fraction, 62% now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in vivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.