Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [³²P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [³H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ∼28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.     

Effects of Amlodipine on Orcadian Rhythms in Blood Pressure,Heart Rate,and Motility: A Telemetric Study in Rats

In male Wistar rats [light (L): 07:00–19:00 h, dark (D): 19:00–07:00 h], the effects of the calcium channel blocker amlodipine (1, 3, 10 mg/kg i.p.) on blood pressure, heart rate, and motor activity were studied by telemetric monitoring. Amlodipine was injected either at 07:00 h or at 19:00 h. Systolic and diastolic blood pressure were dose-dependently decreased with more pronounced effects in the dark span, ED₅₀ values in D were about seven times lower than in L. In contrast, the dose-dependent increase in heart rate was more pronounced in L than in D. No significant effects of amlodipine were found on motor activity. The study gives evidence for a circadian phase-dependency in the cardiovascular effects amlodipine in rats.     

Circadian clock and prothoracicotropic hormone secretion in relation to the larval-larval ecdysis rhythm of the saturniid Samia cynthia ricini

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.     

Adult eclosion rhythm of the Indian meal moth Plodia interpunctella: response to various thermocycles with different means and amplitudes

In addition to photoperiod, thermoperiod (or thermocycle) might be an important Zeitgeber for entraining the circadian oscillator controlling adult eclosion rhythm in the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae). This is confirmed by exposing larvae receiving diapause‐preventing treatments to various thermocycles with different means and amplitudes of temperature. The thermocycles investigated in the present study are TC 8 : 16 h, TC 12 : 12 h, TC 16 : 8 h and TC 20 : 4 h, where T and C represent thermophase (30 °C) and cryophase (20 °C), respectively. For all thermocycles, the peak of adult eclosion rhythm occurs at around the mid‐thermophase. This indicates that the larvae use both ‘temperature‐rise’ and ‘temperature‐fall’ signals to adjust the eclosion phase in each thermocycle. The absence (DD) or presence (LL) of light affects this time‐keeping system slightly under the given thermocycle. The rhythmic adult eclosion noted after exposure of larvae to 30 °C DD for 14 days is recorded in the thermocycles (TC 12 : 12 h, DD; mean temperature = 25 °C) with different amplitudes of 27.5/22.5 °C, 26.5/23.5 °C and 25.5/24.5 °C. The peak in adult eclosion advances in time as the amplitude of the temperature cycle decreases. In the temperature cycle of 25.5/24.5 °C, a peak occurs at the end of the cryophase, 2 h before the temperature‐rise. The adult eclosion rhythm is also observed under various thermocycles (TC 12 : 12 h, DD) consisting of different temperature levels (30 to 20 °C) with different amplitudes. It is found that the temporal position of the peak advances significantly when the amplitude of the thermocycle becomes lower.     

Symbolic dynamics for identifying similarity between rhythms of ecological time series

Explaining the associations between animal populations or between population and environmental signals is an important challenge. The time series that quantify animal populations are often complex, nonlinear, noisy and non‐stationary. These characteristics may make it inappropriate to use traditional techniques when analysing these time series and their mutual dependencies. Here I propose to use symbolic dynamics and techniques from Information Theory to evaluate the degree of dynamic cohesion between time series fluctuations. The main idea is to check whether two (or more) signals tend to oscillate simultaneously, rising and falling together with the same rhythm. Based on synthetic and real time series, I demonstrate that this method is robust to the presence of noise and to the short length of the analysed time series and gives relevant information about the weak relationships between different series. Furthermore, this method appears as simple as classical cross‐correlation and outperforms it in the analysed examples.     

Pyruvate carboxylase in Leptosphaeria michotü. Isolation, properties, intracellular localization and cyclic variations of its activity in relation to polypeptide level

Pyruvate carboxylase (EC 6.4.1.1) was obtained from the fungus Leptosphaeria michotü (West) Sacc. and enriched 543-fold by a 5-step purification procedure as an a4-β4 tetramer of M_r 440000, composedof a M_r 60000 α-subunit, containing bound biotin, and a M_r 50000 β-subunit. The enzyme was active from pH 6.5 to 12.0, with a maximum between pH 8.0 and 8.5. Its specific activity was 125nkat (mg protein)⁻¹: it was not affected by acetyl CoA. A rabbit antiserum raised against the yeast pyruvate carboxylase was specifically reactive against the α-subunits of the L. michotü enzyme. The enzyme was localized into the cytosol by gold-labelled streptavidin and immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Pyruvate carboxylase and acetylCoA carboxylase in L. michotü had synchronous activity rhythms at constant temperature and in darkness; these rhythms were suppressed by cycloheximide or avidin supply. The pyruvate carboxylase level was quantified along the activity rhythm by gel electrophoresis using ³⁵S-streptavidin. and by enzyme-linked immunosorbent assay (ELISA) using serum against the yeast pyruvate carboxylase. The cyclic variations of pyruvate carboxylase activity were correlated with cyclic variations in the enzyme level. Suppression of pyruvate and acetyl CoA carboxylase activities by avidin had a no important effect on the transaminase rhythms of L. michotü .     

Effects of vanadate, N2 and light on the membrane potential of motor cells and the lateral leaflet movements of Desmodium motorium

The lateral leaflets of Desmodium motorium (Houtt.) Merr. exhibit ultradian up- and down movements, which are paralleled by oscillations of the membrane potential of motor cells in the pulvinus. By different treatments we have tested the hypothesis that both that both oscillation-types are causally related. The reactions of the leaflet movement and the membrane potential were evaluated by the following approaches. (1) Application of vanadate. an inhibitor of the proton pump in the plasmalemma. and N₂ suppressed leaflet movements and finally arrested the leaflet in the lower position. Before the oscillations damped out, a strong lengthening in period was found. This indicates that the pump is part of the ultradian clock. A period lenthening and a final suppression of the rhythm by vanadate was also seen in the extracellular electric potential of the pulvinus. Intracellular recordings in situ showed that vanadate application depolarized the motor cells. (2) Light of high fluence rates diminished the amplitude of the oscillations of the membrane potential of single motor cells and shortened the period. The same effects were observed when monitoring the lateral leaflet movement. The leaflet always moved towards the direction of the light. whether it was applied from the abaxial or from the adaxial part of the pulvinus. (3) When light was applied to the pulvinus of lateral leaflets. which had spontancously stopped moving in an upper position. oscillations were induced transiently. This effect was also found for the membrane potential of motor cells in the pulvinus. - Our results thus provide further evidence that the membrane potential controls the volume state of the motor cells in the pulvinus of lateral leaflets of Desmodium motorium .     

Light and temperature entrainment of a locomotor rhythm in honeybees

Abstract. The circadian locomotor (walking) rhythms of forager honeybees (Apis mellifera ligustica L.) were entrained to eight different 24 h light-dark cycles. The phases of activity onset, peak activity, and offset were correlated with the lights-off transition, suggesting lights-off as the primary zeitgeber for the rhythm. Further support for this hypothesis was provided by LD 1:23 experiments, in which entrainment occurred when the light pulse was situated at the end, but not at the beginning, of the subjective photophase. Steady-state entrainment of the locomotor rhythm was achieved with square-wave temperature cycles of 10^oC amplitude under constant dark: most of the activity occurred within the early thermophase. Smaller amplitude temperature cycles yielded relative coordination of the rhythm. Interactions of temperature and light-dark cycles resulted in entrainment patterns different from those elicited in response to either cycle alone or those formed by a simple combination of the two separate responses. Furthermore, temperature cycles having amplitudes insufficient for entrainment of the rhythm nevertheless modified the pattern of entrainment to light - dark cycles, suggesting a synergism of light and temperature effects on the underlying circadian clock system.     

Lack of effect of oxytocin on the numbers of “synaptic” ribbons,cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats

In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied synaptic ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of rat pineals in media containing OT (10^-5 M), noradrenaline (NA, 10^-5 M) or both NA and OT. Pineal glands were derived from rats of three different strains (Sprague-Dawley, Long-Evans and the AVP-deficient strain Brattleboro). Neither morphological nor biochemical analyses showed a difference between control and OT-incubated organs in any of the strains tested. In Brattleboro rats, but not in the other strains, noradrenaline slightly increased the number of SR which was not observed when NA and OT were combined. The addition of NA resulted in distinct augmentation of NAT activity and cGMP content, which were not affected by additional OT application. These results suggest that oxytocin is not crucially involved in the regulation of pineal gland function.