The RNA-binding protein CHLAMY 1 from Chlamydomonas reinhardtii binds specifically to UG (>7) repeat sequences situated in the 3 untranslated regions of several mRNAs. Its binding activity is controlled by the circadian clock. The biochemical purification and characterization of CHLAMY 1 revealed a novel type of RNA-binding protein. It includes two different subunits (named C1 and C3), whose interaction appears necessary for RNA binding. One of them (C3) belongs to the proteins of the CELF (CUG-BP-ETR-3-like factors) family and thus bears three RNA recognition motif domains. The other is composed of three lysine homology domains and a protein-protein interaction domain (WW). The subunits C1 and C3 have theoretical molecular masses of 45 and 52 kDa, respectively, and are present in nearly equal amounts during the circadian cycle. At the beginning of the subjective night, both can be found in protein complexes of 100 to 160 kDa. However, during subjective day when binding activity of CHLAMY 1 is low, the C1 subunit in addition is present in a high-molecular-mass protein complex of more than 680 kDa. These data indicate posttranslational control of the circadian binding activity of CHLAMY 1. Notably, the C3 subunit shows significant homology to the rat CUG-binding protein 2. Anti-C3 antibodies can recognize the rat homologue, which can also be found in a protein complex in this vertebrate.

WOLLNIK, F. AND F. W. TUREK. Estrous correlated modulations of circadian and ultradian wheel-running activity rhythms in LEW!Ztm rats. PHYSIOL BEHAV 43(3) [389][390][391][392][393][394][395][396] 1988.-Estrogen treatment alters the expression ofultradian activity rhythms in male and female LEW/Ztro rats. This finding raises the possibility tbat the expression of ultradian rhythms may vary on different days of the estrous cycle. To test this hypothesis, we recorded the circadian and ultradian wheel-running activity rhythms of entrained (LD 12: 12) and free-running sexually mature LEW/Ztm females during their 4-or 5-day estrous cycle. The mean daily activity, the duration of activity, the circadian period of activity, and the occurrence of ultradian rhythms differed significantly among the days of the estrous cycle. ln LD 12: 12, the phase angle difference between the beginning of activity and light offset varied reliably in 5-day cycling animals. The highest daily mean of activity, the longest duration, and the shortest circadian period length were observed on the day of estrus in both entrained and free-running animals. The day of estrus was characterized by a constant high level of activity throughout the activity phase, while the days following ovulation showed a bi-or trimodal activity pattern. Power spectrum analysis revealed significant ultradian components for the days of metestrus and diestrus, but only circadian components for the days of proestrus and estrus. These results were interpreted as indicating that endogenous changes in circulating hormone levels can induce changes in the ultradian and circadian patterns of wheel-running activity in LEW/Ztm rats. Wheel-running activityEstrous cycle Ultradian and circadian rhythms Laboratory rat STEROID hormones have been reported to modulate the amplitude, phase, and period of mammalian circadian rhythms (for review see [25,30]). In female hamsters and rats, the time of activity onset fluctuates with the estrous cycle in animals entrained to a light-dark cycle as well as in animals maintained under constant illumination or darkness [2,7,14,22]. Activity onset in hamsters occurs earlier on both proestrus and estrus, the days of the estrous cycle when serum levels of estrogen are high [5], whereas in rats the activity onset is earlier only on the day of estrus [2]. Exogenously supplied gonadal hormones also alter the periodicity of circadian rhythms. Silastic estrogen-filled capsules that produce proestrous levels of estrogen shorten the period of the free-running rhythms in ovariectomized rats and hamsters [1,14,31], and increase the total amount of wheelrunning activity [11,22]. Although progesterone alone does not affect circadian rhythms [14,17,22], it can antagonize estrogen's action on activity rhythms in rats and hamsters [3,4,17,22]. It is likely that the interaction of progesterone and estrogen modulates the circadian rhythm of activity in female rats and hamsters on a day-to-day basis [2,22]. Recent studies indicate that gonadal hormones can also alte...

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, we blocked the expression of c-Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c-Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker.

Body temperature of five European hamsters exposed to semi-natural environmental conditions at 47 degrees N in Southern Germany was recorded over a 1.5-year period using intraperitoneal temperature-sensitive radio transmitters. The animals showed pronounced seasonal changes in body weight and reproductive status. Euthermic body temperature changed significantly throughout the year reaching its maximum of 37.9 +/- 0.2 degrees C in April and its minimum of 36.1 +/- 0.4 degrees C in December. Between November and March the hamsters showed regular bouts of hibernation and a few bouts of shallow torpor. During hibernation body temperature correlated with ambient temperature. Monthly means of body temperature during hibernation were highest in November (7.9 +/- 0.8 degrees C) and March (8.2 +/- 0.5 degrees C) and lowest in January (4.4 +/- 0.7 degrees C). Using periodogram analysis methods, a clear diurnal rhythm of euthermic body temperature could be detected between March and August, whereas no such rhythm could be found during fall and winter. During hibernation bouts, no circadian rhythmicity was evident for body temperature apart from body temperature following ambient temperature with a time lag of 3-5 h. On average, hibernation bouts lasted 104.2 +/- 23.8 h with body temperature falling to 6.0 +/- 1.7 degrees C. When entering hibernation the animals cooled at a rate of -0.8 +/- 0.2 degrees C.h-1; when arousing from hibernation they warmed at a rate of 9.9 +/- 2.4 degrees C.h-1. Warming rates were significantly lower in November and December than in January and February, and correlated with ambient temperature (r = -0.46, P < 0.01) and hibernating body temperature (r = -0.47, P < 0.01). Entry into hibernation occurred mostly in the middle of the night (mean time of day 0148 hours +/- 3.4 h), while spontaneous arousals were widely scattered across day and night. For all animals regression analysis revealed free-running circadian rhythms for the timing of arousal. These results suggest that entry into hibernation is either induced by environmental effects or by a circadian clock with a period of 24 h, whereas arousal from hibernation is controlled by an endogenous rhythm with a period different from 24 h.

In our studies on diurnal 6-sulphatoxymelatonin (aMT6s) rhythms in various species, we have sometimes obtained fluctuating patterns. In most of these, the volume of individual urine fractions was not accurately measured because of methodological problems. Here, we report a simple method to overcome these problems by using urinary creatinine to estimate urine volume. The benefit of this method is demonstrated in two representative examples of the diurnal aMT6s rhythms of rats, domestic pigs and humans. Because the human urine fractions were collected accurately, the qualitative pattern of the aMT6s rhythm was not altered by using urinary creatinine as a substitute for urine volume. The total creatinine excretion (urine volume x creatinine concentration) was constant within a small range and showed no diurnal rhythm. In rats and pigs, the highly variable aMT6s concentrations relative to urine volume throughout the 24-hr period were changed drastically by referring to creatinine. All aMT6s patterns became stable and qualitatively similar to those of the rest of the group. From these results it can be concluded that creatinine is an adequate substitute for urine volume and a beneficial parameter with which to overcome technical problems with urine collection from laboratory animals or unknown urine volumes in human studies.

1. To investigate the role of the Jun transcription factors in neuronal differentiation, programmed neuronal cell death, and neuronal plasticity, we used phosphorothioate oligodeoxynucleotides (S-ODN) to inhibit selectively the expression of c-Jun, JunB, and JunD. 2. We have shown previously that in contrast to c-Jun, the JunB and JunD transcription factors are negative regulators of cell growth in various cell lines. Here we confirm this finding in primary human fibroblasts. 3. c-Jun and JunB are counterplayers not only with respect to proliferation, but also in cell differentiation. Since JunB expression is essential for neuronal differentiation, we analyzed possible posttranslational modifications of JunB after induction of PC-12 cell differentiation by nerve growth factor (NGF). 4. JunB was strongly phosphorylated after induction of PC-12 cell differentiation with NGF but not after stimulation of cell proliferation with serum. Thus, while cell proliferation is associated with c-Jun phosphorylation, cell differentiation is correlated with JunB phosphorylation. This supports the finding that c-Jun and JunB play antagonistic roles in both proliferation and differentiation. 5. The JunB transcription factor together with the c-Fos transcription factor is also induced in vivo in the suprachiasmatic nucleus (SCN) of rat brain after a light stimulus that induces resetting of the circadian clock. 6. Using antisense oligonucleotides injected into the third ventricle, we selectively cosuppressed the two transcription factors in vivo as shown by immunohistochemistry. Expression of c-Jun, JunD, and FosB was not affected. Inhibition of JunB and c-Fos expression prevented the light-induced phase shift of the circadian rhythm. In contrast, rats injected with a randomized control oligonucleotide showed the same phase shift as untreated animals. 7. In primary rat hippocampal cultures, anti-c-jun S-ODN selectively inhibited neuronal cell death and promoted neuronal survival. This indicates a causal role of c-Jun in programmed neuronal cell death. 8. These findings demonstrate the essential role of inducible transcription factors in the reprogramming of cells to a different functional state. Jun transcription factors play an essential role not only in fundamental processes such as cell proliferation, differentiation, and programmed neuronal cell death, but also in such complex processes as plastic adaptations in the mature brain. The inhibition of neuronal cell death by anti-c-jun S-ODN shows the great therapeutic potential of selective antisense oligonucleotides.

Spontaneous locomotor activity (LA) was recorded in five inbred strains of laboratory rats (ACI/ZtmKEY WORDS: circadian and ultradian rhythms; strain differences; locomotor activity; laboratory rat.

. Effects of adult or perinatal hormonal environment on ultradian rhythms in locomotor activity of laboratory LEWIZtm rats. PHYSIOL BEHA V 38(2) [229][230][231][232][233][234][235][236][237][238][239][240] 1986.-Four experiments were performed with male and female rats of the inbred strain LEW/Ztm maintained under a light-dark schedule of 12: 12 hours. The animals were subject to castration CGOX) or ovariectomy (OVX), estradiol 17J3-implantation (E2-capsules), and perinatal hormonal treatments with testosterone propionate (TP) aod an androgen antagonist (cyproterone acetate, CA). Results indicated a difference in the locomotor activity pattern between the two sexes as a result of the endogenous estradiol levels of the adult animals. The activity pall ern of male LEW rats was characterized by ultradian rhythms of 4 and 4.8 hr periods. The female LEW rats, on the other hand, generally exhibited a clear circadian activity pattern and no uttradian activity rhythms. Following ovariectomy, each of the females showed distinct ultradian rhythms. These disappeared after E2-implantation. Castration of adult males had no effect on the ultradian activity pattern. Implantation of E2-capsules resulted in a marked decrease of the ultradian activity components. Perinatal treatment of the males with an androgen antagonist (CA) did not appear to effect ultradian rhythms during adulthood. Females treated perinatally with testosterone showed a significant increase in the ultradian activity components. This effect is assumed to be due to low estrogen levels in these animals during adulthood. Our study supports the assumption that ultradian rhythms are a result of changes in I he phase relationships between several circadian oscillators. The synchrony of these oscillations seems to be facilitated by estradiol. Ultradian rhythmsLocomotor activity Gonadectomy Brain differentiation Laboratory rat SEVERAL weU-known phenomena of circadian rhythms in mammals can be explained by the model of the circadian system as a composite of multiple, highly ordered oscillators [40][41][42]46]located in or close to the suprachiasmatic nuclei in the hypothalamus [47]. The origin of similar temporal variations on a smaUer time scale, caUed ultradian or short-term rhythms, remains unclear [11]. Ultradian rhythms in locomotor activity are known to exist in laboratory rats [8, 9, 2~30, 49], however, attracting much less attention than circadian rhythms.A recent study of LEW/Ztm rats has shown that ultradian rhythms of 4 and 4.8 hr periods are geneticaUy fixed in this Estradiol Testosterone inbred strain (9]. The persistence of these ultradian rhythms foUowing the disruption of circadian organization under continuous light supported the assumption that the activity rhythms of this strain are caused by an independent ultradian oscillator [9]. Alternatively, the persistence of ultradian rhythms can be explained by changes in the phase relationships between several circadian oscillators [46). The ultradian rhythms of the inbred strain LEW/Ztm are sex-specific, ...