Fructose 2, 6-Bisphosphate and Circadian Rhythmicity

We were able to demonstrate the presence of F 2,6-BP in Acetabularia in 7 out of 7 experiments. The amount varies between 4 and 38 pmole par mg protein. We were not able to evidence a circadian rhythm (CR) in its content. However, important fluctuations occur .(Fig. 1). This, of course excludes any precise conclusion about absolute amounts. Biologically active substances often exert an action modulated by circadian time. Thus, the effect of exogenous F 2,6-BP was assayed by fragmenting the long cell in F 2,6-BP-containing sea-water, and then follow growth and cap formation (we performed the experiment at different times during the 24 h cycle, in LD 12:12 conditions. Interestingly, the growth curves (obtained with 4 different concentrations) are statistically accelerated when the treatment had been performed at the beginning of the 24 h cycle (circadian time, CT, 0 is the transition time dark/light), less at CT 9.5, nul at CT 12 and again significant at CT 20. (Fig.IV). There is apparently no strictly defined light effect that could immediately modify the F-2,6-BP level, but there is presumably an important influence of CT-dependent physiological state of the alga. Again, it should be underlined that experimental biology should take time into account.     

Photic phase response curve in Octodon degus: assessment as a function of activity phase preference

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.     

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.     

褪黑素对大鼠下丘脑薄片视交叉上核神经元放电昼夜节律性的调制

先用持续光照和松果腺切除预处理大鼠,然后制成下丘脑薄片,记录其视交叉上核（ＳＣＮ）神经元自发放电,观察其昼夜变化和褪黑素（ＭＥＬ）对它的影响。实验结果表明：⑴在正常光照（光照：黑暗＝１２：１２）条件下,ＳＣＮ神经元自发放电频率呈现昼夜低的节律性。在昼夜时间（ＣＴ）６－８出现放电高峰,频率约为８．３Ｈｚ;在ＣＴ１８－２０出现低谷,频率约为３．８Ｈｚ。松果腺切除后,ＳＣＮ神经元自发放电的昼夜节律性基本     

Circadian Intraocular Pressure Rhythms in Athletic Horses under Different Lighting Regime

The present study was undertaken to investigate the existence of intraocular pressure (IOP) rhythms in athletic thoroughbred horses maintained under a 24 h cycle of light and darkness (LD) or under constant light (LL) or constant dark (DD) conditions. We identified an IOP circadian rhythm that is entrained to the 24 h LD cycle. IOP was low during the dark phase and high during the light phase, with a peak at the end of the light phase (ZT10). The circadian rhythm of IOP persisted in DD (with a peak at CT9.5), demonstrating an endogenous component in IOP rhythm. As previously shown in other mammalian species, horse IOP circadian rhythmicity was abolished in LL. Because tonometry is performed in horses for the diagnosis of ophthalmologic diseases, such as glaucoma or anterior uveitis, the daily variation in IOP must be taken into account in clinical practice to properly time tests and to interpret clinical findings.     

Influence of fructose 2,6-bisphosphate and MgATP on rat liver phosphofructokinase at pH 7: evidence for a complex interdependence.

The relationship between fructose 2,6-bisphosphate (Fru-2,6-BP) activation and MgATP inhibition of rat liver phosphofructokinase has been comprehensively evaluated at pH 7. When either ligand is varied at a fixed concentration of the other, its influence on the concentration of fructose 6-phosphate (Fru-6-P) required to produce half-maximal velocity, Ka, is usually well described by the same simple, single-modifier linkage expression that described the actions of these ligands at pH 9. However, the effects of both ligands together cannot be described by the same overall linkage relationship that described their actions at pH 9. Specifically, despite an overall antagonistic relationship between the binding of MgATP and that of Fru-2,6-BP, very low concentrations of Fru-2,6-BP appear to facilitate the binding of MgATP to an appreciable degree. Also, MgATP at high concentration appears to inhibit the binding of Fru-2,6-BP to a significantly greater extent than its actions at lower concentration would predict. These additional features of MgATP-Fru-2,6-BP interaction have been incorporated into an overall linkage expression describing the actions of both MgATP and Fru-2,6-BP on Ka for Fru-6-P. The best fit parameters predict the data to within an average standard error of +/- 21%.     

Influence of fructose 2,6-bisphosphate on the aggregation properties of rat liver phosphofructokinase

The influence that fructose 2,6-bisphosphate (Fru-2,6-BP) has on the aggregation properties of rat liver phosphofructokinase has been studied by observing the fluorescence polarization of the enzyme covalently bound to the fluorescent probe pyrenebutyric acid. Fru-2,6-BP dramatically slows the dissociation of the high molecular weight aggregate forms of the enzyme when the enzyme is diluted to 3.2 micrograms/ml (4 X 10(-8) M subunits). Furthermore, Fru-2,6-BP is a strong promoter of reassociation to tetramer and larger forms if the enzyme has been previously allowed to dissociate to the dimer in its absence. Unlike many other positive effectors of liver phosphofructokinase, Fru-2,6-BP is also able to overcome the tendency of MgATP to promote tetramer formation and instead stabilize a very high degree of high molecular weight aggregate formation even in the presence of MgATP. The apparent affinity of liver phosphofructokinase for Fru-2,6-BP was measured by its ability to promote reassociation and compared to that for Fru-1,6-BP. The apparent dissociation constant for Fru-2,6-BP under these conditions is 36 microM, about 40-fold lower than the value of 1.4 mM measured for Fru-1,6-BP. Both ligands demonstrate synergism with the substrate Fru-6-P, which can lower the dissociation constant for Fru-2,6-BP 9-fold to 4 microM and that for Fru-1,6-BP 5-fold to 0.28 mM. These data are interpreted to suggest that influencing the aggregation state of rat liver phosphofructokinase may be one way in which Fru-2,6-BP achieves its effects on the enzyme in vivo.     

The effects of low-dose 0.5-mg melatonin on the free-running circadian rhythms of blind subjects

Exogenous melatonin (0.5-10 mg) has been shown to entrain the free-running circadian rhythms of some blind subjects. The aim of this study was to assess further the entraining effects of a daily dose of 0.5 mg melatonin on the cortisol rhythm and its acute effects on subjective sleep in blind subjects with free-running 6-sulphatoxymelatonin (aMT6s) rhythms (circadian period [tau] 24.23-24.95 h). Ten subjects (9 males) were studied, aged 32 to 65 years, with no conscious light perception (NPL). In a placebo-controlled, single-blind design, subjects received 0.5 mg melatonin or placebo p.o. daily at 2100 h (treatment duration 26-81 days depending on individuals' circadian period). Subjective sleep was assessed from daily sleep and nap diaries. Urinary cortisol and aMT6s were assessed for 24 to 48 h weekly and measured by radioimmunoassay. Seven subjects exhibited an entrained or shortened cortisol period during melatonin treatment. Of these, 4 subjects entrained with a period indistinguishable from 24 h, 2 subjects continued to free run for up to 25 days during melatonin treatment before their cortisol rhythm became entrained, and 1 subject appeared to exhibit a shortened cortisol period throughout melatonin treatment. The subjects who entrained within 7 days did so when melatonin treatment commenced in the phase advance portion of the melatonin PRC (CT6-18). When melatonin treatment ceased, cortisol and aMT6s rhythms free ran at a similar period to before treatment. Three subjects failed to entrain with initial melatonin treatment commencing in the phase delay portion of the PRC. During melatonin treatment, there was a significant increase in nighttime sleep duration and a reduction in the number and duration of daytime naps. The positive effect of melatonin on sleep may be partly due to its acute soporific properties. The findings demonstrate that a daily dose of 0.5 mg melatonin is effective at entraining the free-running circadian systems in most of the blind subjects studied, and that circadian time (CT) of administration of melatonin may be important in determining whether a subject entrains to melatonin treatment. Optimal treatment with melatonin for this non-24-h sleep disorder should correct the underlying circadian disorder (to entrain the sleep-wake cycle) in addition to improving sleep acutely.     

Characteristics of a circadian pacemaker in the suprachiasmatic nucleus

Summary The nature of the circadian rhythms of the SCN in a hypothalamic island was examined in male rats by recording multiple unit activity from the SCN for longer durations. Successful continuous recording lasted up to 35 days. Neural activity of the SCN inside the island showed free-running rhythms whose periods were slightly longer than 24 h (Figs. 2, 3, Table 1). When the retino-hypothalamic pathway was spared, re-entrainment to a displaced light and dark cycle was attained following a transition period of a few days (Fig. 4). Phases of the rhythms shifted in a phase-dependent manner in response to single light pulses interrupting constant darkness (Fig. 5 and Fig. 6). These results suggest an endogenous nature of the circadian rhythm of the SCN within the hypothalamic island. Thus, neurons or neuronal networks in the SCN may have not only an inherent ability to generate a circadian rhythm, but also an intricate machinery to regulate its phase. Simultaneous recordings from the left and right SCN showed a slight but visible discrepancy in their phases between the two rhythms in 3 out of 12 cases (Fig. 7).Abbreviations LL constant light - LD light-dark - DD constant darkness - SCN Suprachiasmatic nucleus     

Characteristics of the Light-Induced Phase Response of Circadian Activity Rhythms in Common Marmosets,Callithrix j. jacchus [Primates-Cebidae]

Phase-response experiments using 1-h light pulses (LPs) of 1,100 lux applied under constant dim light of 0.3 lux were conducted with common marmosets, Callithrix j. jacchus, in order to obtain a complete phase-response curve established according to the common experimental procedure in a diurnal primate. Maximal phase delays of the free-running circadian activity rhythm (- 90 min) were induced by LPs delivered at circadian time (CT) 12; e.g., during the beginning of the marmosets' rest time, maximal advances (+ 25 min) were elicited by pulses administered during the late subjective night at CT 21. In contrast to rodents, neither regular transient cycles nor regular period responses resulted from LP applications at different phases. To check whether the underlying period length affects the phase response in primates as well, the marmosets' circadian timing system was entrained to 25 h by a lightrdark (LD) cycle of 12.5:12.5 h. The 1-h LPs were delivered during the first circadian cycle produced under constant dim light after the entraining LD periods. Here, LPs applied at CT 21 led to phase advances exceeding those measured during the steady-state free run. At CT 12, minor or no phase delays could be elicited. These findings show that the phase-shifting effect of LPs on the circadian system of marmosets is similar to that observed in other diurnal mammals. Some of the results indicate that in this diurnal primate, LP-induced phase shifts may be mediated in part by a light-induced increase in locomotor activity (arousal).     

Characteristics of the Light-Induced Phase Response of Circadian Activity Rhythms in Common Marmosets, Callithrix j. jacchus [Primates-Cebidae]

Phase-response experiments using 1-h light pulses (LPs) of 1,100 lux applied under constant dim light of 0.3 lux were conducted with common marmosets, Callithrix j. jacchus, in order to obtain a complete phase-response curve established according to the common experimental procedure in a diurnal primate. Maximal phase delays of the free-running circadian activity rhythm (- 90 min) were induced by LPs delivered at circadian time (CT) 12; e.g., during the beginning of the marmosets' rest time, maximal advances (+ 25 min) were elicited by pulses administered during the late subjective night at CT 21. In contrast to rodents, neither regular transient cycles nor regular period responses resulted from LP applications at different phases. To check whether the underlying period length affects the phase response in primates as well, the marmosets' circadian timing system was entrained to 25 h by a lightrdark (LD) cycle of 12.5:12.5 h. The 1-h LPs were delivered during the first circadian cycle produced under constant dim light after the entraining LD periods. Here, LPs applied at CT 21 led to phase advances exceeding those measured during the steady-state free run. At CT 12, minor or no phase delays could be elicited. These findings show that the phase-shifting effect of LPs on the circadian system of marmosets is similar to that observed in other diurnal mammals. Some of the results indicate that in this diurnal primate, LP-induced phase shifts may be mediated in part by a light-induced increase in locomotor activity (arousal).     

The early snail acquires the learning. Comparison of scores for conditioned taste aversion between morning and afternoon

The pond snail Lymnaea stagnalis acquires conditioned taste aversion (CTA) and maintains its memory for more than a month. Snails in our laboratory were cultured at 20 degrees C on a 12:12 light-dark cycle (light from 7 am to 7 pm). To examine the hours during which snails acquire CTA effectively, we trained some snails in the morning and others in the afternoon, and then compared their scores. CTA developed in both cases, but scores were significantly better in the morning than in the afternoon. To elucidate the cause of this difference in scores, we observed the voluntary activity of snails and found the circadian rhythm reflected in the snails' free-movement distances; distances at the circadian time 0-12 (daytime) were significantly longer than those at the circadian time 12-24 (nighttime). This rhythm was kept up for at least 3 days, even in constant darkness. In conclusion, L. stagnalis should be trained in the morning to acquire associative learning, possibly because of its greater propensity to roam about at that time as opposed to the afternoon.     

Photoperiodic ovarian response in yellow-throated sparrow, Gymnorhis xanthocollis: involvement of circadian rhythm

The experiments aim to investigate the mechanism of photoperiodic time measurement during photoperiodic ovarian response of subtropical yellow-throated sparrow. Groups of the photosensitive female birds were exposed to various night-interruption cycles for a period of 35 days. These light-dark cycles consisted of a basic photophase of 6h and 1h photointerruption of the 18h dark phase in 24h cycle at different points. A control group was also placed under 7L/17D. Ovarian response was observed in the night-interruption cycles in which the photointerruption of dark phase was made 12h after the onset of basic photophase. The results are consistent with the Bünning hypothesis and indicate that an endogenous circadian rhythm is involved in photoperiodic time measurement during initiation of ovarian growth in this species.     

Photic and nonphotic circadian phase resetting in a diurnal primate, the common marmoset

Despite the considerable literature on circadian entrainment, there is little information on this subject in diurnal mammals. Contributing to this lack of understanding is the problem of separating photic from nonphotic (behavioral) phase-resetting events in diurnal species. In the present study, photic phase resetting was obtained in diurnal common marmosets held under constant dim light (DimDim; <0.5 lx) by using a 20-s pulse of bright light to minimize time available for behavioral arousal. This stimulus elicited phase advances at circadian time (CT) 18-22 and phase delays at CT9-12. Daily presentation of these 20-s pulses produced entrainment with a phase angle of approximately 11 h (0 h = activity onset). Nonphotic phase resetting was obtained under DimDim with the use of a 1-h-induced activity pulse, consisting of intermittent cage agitation and water sprinkling, delivered in total darkness to minimize photic effects. This stimulus caused phase delays at CT20-24, and entrainment to a scheduled daily regimen of these pulses occurred with a phase angle of approximately 0 h. These results indicate that photic and nonphotic phase-response curves (PRCs) of marmosets are similar to those of nocturnal rodents and that nonphotic PRCs are keyed to the phase of the suprachiasmatic nucleus pacemaker, not to the phase of the activity-rest cycle.     

Evidence of circadian rhythm of electric discharge in Eigenmannia virescens system

The gymnotid electric fish, Eigenmannia virescens, exhibits electric discharge rhythmicity both in alternate light-dark (LD; 12h light, 12h dark [LD 12:12]) and in constant dark (DD) conditions. It suggests that the electric discharge rhythm is under control of the circadian clock. The free-running periods (FRPs) of electric discharge rhythms at 21°C in DD are greater than, but close to, 24h. The maximum of the electric discharge in the Eigenmannia system peaks approximately at circadian time 6 (CT6) in the middle of the subjective day. The circadian oscillator in the system is temperature compensated. This original report reveals the relationship between electric discharge activity and the circadian pacemaker in Eigenmannia and provides an alternative system to investigate circadian rhythms in vertebrates. (Chronobiology International, 17(1), 43-48, 2000)     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

Phase and period responses to short light pulses in a wild diurnal rodent,Funambulus pennanti

Photic phase response curves (PRCs) have been extensively studied in many laboratory-bred diurnal and nocturnal rodents. However, comparatively fewer studies have addressed the effects of photic cues on wild diurnal mammals. Hence, we studied the effects of short durations of light pulses on the circadian systems of the diurnal Indian Palm squirrel, Funambulus pennanti. Adult males entrained to a light–dark cycle (12?h–12?h) were transferred to constant darkness (DD). Free-running animals were exposed to brief light pulses (250 lux) of 15?min, 3 circadian hours (CT) apart (CT 0, 3, 6, 9, 12, 15, 18 and 21). Phase shifts evoked at different phases were plotted against CT and a PRC was constructed. F. pennanti exhibited phase-dependent phase shifts at all the CTs studied, and the PRC obtained was of type 1 at the intensity of light used. Phase advances were evoked during the early subjective day and late subjective night, while phase delays occurred during the late subjective day and early subjective night, with maximum phase delay at CT 15 (?2.04?±?0.23?h), and maximum phase advance at CT 21 (1.88?±?0.31?h). No dead zone was seen at this resolution. The free-running period of the rhythm was concurrently lengthened (deceleration) during the late subjective day and early subjective night, while period shortening (acceleration) occurred during the late subjective night. The maximum deceleration was noticed at CT 15 (?0.40?±?0.09?h) and the maximum acceleration at CT 21 (0.39?±?0.07?h). A significant positive correlation exists between the phase shifts and the period changes (r?=?0.684, p?=?0.001). The shapes of both the PRC and period response curve (τRC) qualitatively resemble each other. This suggests that the palm squirrel’s circadian system is entrained both by phase and period responses to light. Thus, F. pennanti exhibits robust clock-resetting in response to light pulses.