Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

The function of circadian RNA-binding proteins and their cis-acting elements in microalgae

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin-binding protein (LBP) is controlled at the translational level. Thereby, a clock-controlled RNA-binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG-repeat, which is situated in the lbp 3^' UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3^' UTRs of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG-repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO₂ metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.     

IMMUNOLOCALIZATION OF NITRATE REDUCTASE IN THE MARINE DINOFLAGELLATE GONYAULAX POLYEDRA (PYRROPHYTA)1

A polyclonal antibody, raised against nitrate reductase (NR) purified from the photosynthetic dinoflagellate Gonyaulax polyedra Stein, was used as a probe in immunogold-labeling experiments on thin sections prepared from cells harvested both during day and night phases. Previous experiments have shown that both NR activity and the amount of immunoreactive NR in cell extracts is greater when day-phase cells are examined, and this property was exploited as an internal control for the cytochemical labeling. We observed that in day-phase cells, chloroplasts contained approximately three times more gold particles than night-phase cells (highly significant difference; P < 0.0001), whereas cytoplasmic labeling levels remained relatively level between day and night. We conclude from the diurnal difference in labeling that our antibody faithfully reflects the distribution of NR in Gonyaulax cells. Thus, as in to some other higher plants and green algae, Gonyaulax compartmentalizes active NR in its chloroplasts.     

The Function of Circadian RNA‐Binding Proteins and Their cis‐Acting Elements in Microalgae

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin‐binding protein (LBP) is controlled at the translational level. Thereby, a clock‐controlled RNA‐binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG‐repeat, which is situated in the lbp 3^′ UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock‐controlled RNA‐binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3^′ UTRs of several mRNAs and recognizes them all via a common cis‐acting element, composed of at least seven UG‐repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO₂ metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.     

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

The effect of repeated pulses of light at the same time on period responses of the rat circadian pacemaker

A consequence of simple velocity-based models is that, in response to light pulses, the circadian period should adjust inversely to phase. In addition, because of the interaction of circadian period and phase response, earlier circadian period changes should modify later circadian period changes. The literature contains few mentions of response curves of circadian period responses following light pulses. Rats were exposed to four pulses of light (60 minutes, 1000 lux) at the same circadian time, a minimum of 26 days apart; we assessed period responses and possible bias in the period-response curve. Modulation of circadian period following light-induced phase responses was examined by assessing the period of running wheel activity onset. Phase and circadian period were not consistently found to share an inverse relationship. Moreover, biases in initial period tended to be increased by the experimental protocol regardless of circadian time of pulse. Rats with a short initial (high-velocity) period had a lengthened period, while rats with a long initial period (low velocity) tended to have a reduce period. However, rats with a long initial period were phase delay biased, not phase advance biased. These results do not support a simple velocity model of the pacemaker. (Chronobiology International, 18(2), 187-201, 2001)     

Rapid Phase Resetting of a Mammalian Orcadian Rhythm by Brief Light Pulses

The phase-shift (Δψ) responses of the circadian rhythm in the field mouse Mus booduga to brief light pulses (LPs) of 15 minutes duration and 1000 lux intensity were measured in 90 experiments. In each experiment, a resetting light pulse LP₁ was administered at CT14 (CT, circadian time), and a scanning light pulse LP₂ was then variously administered in separate experiments at CT16, CT20, and CT22 in the same and in the next circadian cycle. The Δψ obtained in all these two-pulse experiments did not differ significantly from theoretical values computed on the assumption that LP₁ reset the phase response curve (PRC) rapidly. In each case, the steady-state Δψ observed after LP₁ and LP₂ differed significantly from the Δψ obtained at the same CT in determination of the single-pulse PRC (control) and also differed significantly from the values on the assumption of no Δψ in the PRC following LP₁. These results indicate that the circadian pacemaker of M. booduga, as measured by its PRC, is substantially reset within 2h after a light pulse at CT14. (Chronobiology International 14(6), 537–548, 1997)     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411-421, 1996)     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411–421, 1996)     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

Low,but not high,doses of melatonin entrained a free-running blind person with a long circadian period

In a previous report, we were unable to entrain one out of seven totally blind people with free-running endogenous melatonin rhythms to 10 mg of exogenous melatonin. This person had the longest circadian period (24.9 h) of the group. We now find that this person can be entrained to 0.5 mg of melatonin, but not to 20 mg. These results are consistent with the idea that too much melatonin may spill over onto the wrong zone of the melatonin phase–response curve.     

Zeitgeber hierarchy in humans: resetting the circadian phase positions of blind people using melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free-running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.     

Zeitgeber Hierarchy in Humans: Resetting the Circadian Phase Positions of Blind People Using Melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free‐running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.     

Circadian and light-induced conductance changes in putative pacemaker cells of Bulla gouldiana

The ocular circadian rhythm of compound action potential frequency in Bulla gouldiana is driven by rhythmic changes in the membrane potential of putative circadian pacemaker cells. Changes in the membrane potential of these neurons is required for light-induced phase shifts of the rhythm. We have tested the proposition that these changes in membrane potential reflect underlying changes in ionic conductances. We have found that: 1. Membrane conductance in the dark is highest during the subjective night when the cells are hyperpolarized, decreases as the cells depolarize spontaneously near projected dawn and is lowest during the subjective day. The changes in membrane potential and conductance follow a similar time course. 2. Long pulses of light delivered to eyes during their subjective night produce a characteristic response: There is initially a large, phasic depolarization accompanied by a burst of CAPs; this is followed by a repolarizing phase during which CAP activity is reduced to zero; and finally a tonic depolarization develops that is accompanied by a resumption of CAP activity at a steady rate. 3. During the subjective night, the tonic depolarization is accompanied by a decrease in conductance compared to the previous dark value. However, light pulses of similar duration delivered to eyes during their subjective day causes tonic depolarizations and increased CAP activity, but no measurable change in conductance. 4. Membrane responses to light are sensitive to agents that reduce Ca2+ flux. Light pulses during the subjective night produce a phasic depolarization, but the repolarization phase is eliminated in low Ca2+/EGTA seawater and is reduced in 5 mM Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photobiology of the Gonyaulax circadian system. I. Different phase response curves for red and blue light

Phase responses to red and blue light pulses were measured at different times during the circadian cycle (phase response curves, PRC) in the marine unicellular dinoflagellate Gonyaulaxpolyedra Stein. Pulses were given during a 24-h period of darkness; thereafter, cultures were released into constant dim red light for the assessment of phase and period. The results confirmed earlier findings that the Gonyaulax circadian system receives light signals via two distinct input pathways. During the subjective day and for the first 3 h of the subjective night, red and blue light pulses led to identical phase responses. For the rest of the circadian cycle, however, phase responses to pulses of either red or blue light differed drastically both in their amplitude and direction (advances or delays). Thus, the Gonyaulax light PRC is generated by two distinct light responses. One of these represents responses via a light input that is responsive both to red and blue light mainly producing small delays. The other represents responses of a primarily blue-sensitive input system leading to large advances restricted to the subjective night. Via feed-back, the blue-sensitive light input appears to be under the control of the circadian system. Received: 27 November 1996/Accepted: 30 January 1997     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.