Control Of Circadian Rhythms Research Articles

Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.

MPer1 and mPer2 mutant mice show regular spatial and contextual learning in standardized tests for hippocampus-dependent learning

Learning and memory, like most physiological processes, seem to be under the control of circadian rhythm. The recently cloned mPer1 and mPer2 genes play an important role in the regulation of the circadian rhythm. In this study, we tested mPer1 and mPer2 mutant mice in two different learning and memory paradigms, a water-maze place navigation task and contextual fear conditioning. In both learning tests, the hippocampus is critically involved. None of these learning types were affected by the mutations, suggesting that mPer1 and mPer2 do not play a major role in the regulation of hippocampus-dependent learning and memory.

Control of Mammalian Circadian Rhythm by CKIε-Regulated Proteasome-Mediated PER2 Degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

Molecular Biology of Circadian Rhythms

Amita Sehgal (Ed.) Wiley-Liss, 2004. $89.95, 283 pp. ISBN 0–471–41824–2 At the intersection of Genes, Brain and Behavior are Time, Love and Memory (Weiner 1999). The study of the genetic basis of these ‘simple’ behaviors, championed by Seymour Benzer in the 1960s and early 1970s, has paid rich dividends. The greatest progress has been made in understanding the molecular basis for circadian rhythms. Tremendous advances have been made in understanding the molecular feedback loops that underlie the capacity of organisms to measure the 24 h day and anticipate the next dawn. Painstaking work in the 1970s and 1980s led to the identification of mutants affecting circadian timing in model organisms, but as recently as 10 years ago, there were only two identified genes, period in Drosophila and frequency in Neurospora, that were routinely accepted as ‘clock genes’, genes essential for proper circadian timekeeping. The 1990s was a decade of explosive growth in the field, as workers exploited ‘new’ molecular methods including the polymerase chain reaction and genomic databases, combined with the faithful standby, the mutagenesis screen. As molecular information emerged, the similarity in the molecular basis of clocks in Drosophila and mammalian systems became apparent, fueling further advances. Not to be outdone, our colleagues studying organisms without brains (prokaryotic cyanobacteria, plants and bread mold) also made tremendous strides. While a review of the molecular bases for circadian clocks fit quite nicely into a 20-page review 5 years ago (Dunlap 1999), the expansion in the area has created a need for a text such as the one edited by Amita Sehgal. The volume by Sehgal and colleagues is aimed at an advanced undergraduate/graduate student level. The introductory chapter introduces concepts and terminology. In my view, portions of the opening three chapters of another recent text (Dunlap et al. 2003) provide a much more readable, clear and well-illustrated introduction to the field. The second chapter of this volume reviews methods to identify or study circadian genes and is likely essential for the target audience. The core of the book consists of six chapters devoted to describing the current understanding of the molecular control of circadian rhythms in animal models (Drosophila, non-mammalian vertebrates and mammals) and in cyanobacteria, Neurospora and plants. These chapters present useful information about the methods for studying each of these organisms and review the mutants and genes involved in the clockwork of each species. Many illustrations are stylized versions of data from published papers but often without providing a citation to the original source. Indeed, the chapters average only 15 references each; those encouraging students to pursue the primary literature will find this book dissatisfying. The illustrations and one-stop shopping convenience of this volume make it a useful complement to detailed, well-referenced reviews published by others (Allada et al. 2001; Johnson 2001; Loros & Dunlap 2001; Reppert & Weaver 2002; Williams & Sehgal 2001; compendia of review articles have also appeared recently in Cell and Tissue Research[volume 309, 2002] and in Novartis Foundation Symposia[volume 253, 2003]). The final section of the book is three chapters on ‘circadian organization in complex organisms’ that discuss selected physiological aspects of rhythmicity. These chapters do not appear to be well integrated with the remainder of the book or each other. The book includes a reasonable but not exhaustive index. As acknowledged by Dr Sehgal in the foreword, authors of several of the chapters have no direct experience working with the organism that they write about. The authors seem to have been selected for current or past research or institutional affiliation rather than on expertise with the specific model organism. To their credit, however, these authors have gone to tremendous effort to bring the information together, and indeed I believe that some chapters benefit from taking advantage of the opportunity to bring together and illustrate old and newer information, rather than being a revision of a review written by a more recognizable name. Overall, this volume does a good job of providing a timely snapshot of the molecular mechanisms of biological timekeeping as we understand it today and will be a good introduction to the topic for those interested in how genes and their products control rhythmic physiology and behavior. Experts in the field may have quibbles with specific points and with the emphasis of some chapters, but the book succeeds in its aim for its target audience.

Differential control of peripheral circadian rhythms by suprachiasmatic-dependent neural signals

Although dependent on the integrity of a central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN), endogenous daily (circadian) rhythms are expressed in a wide variety of peripheral organs. The pathways by which the pacemaker controls the periphery are unclear. Here, we used parabiosis between intact and SCN-lesioned mice to show that nonneural (behavioral or bloodborne) signals are adequate to maintain circadian rhythms of clock gene expression in liver and kidney, but not in heart, spleen, or skeletal muscle. These results indicate that the SCN regulates expression of circadian oscillations in different peripheral organs by diverse pathways.

Circadian-Associated Rice Pseudo Response Regulators (OsPRRs): Insight into the Control of Flowering Time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.

Serotonin 5-HT7 Receptor Antagonists

The 5-HT7R is the most recent addition to the burgeoning family of serotonin receptors. Preliminary evidences suggest that it may be involved in depression, control of circadian rhythms, and relaxation in a variety of vascular smooth muscles, indicating the high potential of 5-HT7R ligands as new therapeutic drugs. During the last four years several selective 5-HT7R antagonists have been discovered, and we have recently contributed to this field with the definition of a pharmacophoric hypothesis for 5-HT7R antagonism and a computational model of ligand-receptor interaction of new naphtholactam and naphthosultam derivatives acting at this receptor. This article will review the development of 5-HT7R antagonists with an emphasis on selective antagonists, their structural requirements and ligand-receptor interactions, as well as the potential therapeutic opportunities surrounding 5-HT7R ligands. Keywords: serotonin, 5-ht7 receptor antagonists, pharmacophore model, computational model, ligand-receptor interaction

Diurnal changes of tonic nociceptive responses in mice: evidence for a proalgesic role of melatonin

Diurnal variations in tonic pain reactions have been described in mice tested in Spring, but the underlying mechanisms are still unknown. We tested the potential role of melatonin, a key hormone in the control of neuro-endocrine circadian rhythms. The experiments were performed in male CBA/J mice housed under controlled temperature, humidity, and light (12/12 dark/light cycle) conditions, during the Light (7–10 a.m.) or Dark (7–10 p.m.) phases of the diurnal cycle. In a first group of experiments, animals were either pretreated with i.p. saline (controls) or with the melatonin receptor antagonist, luzindole (30 mg/kg), before the s.c. injection of a dilute formalin solution into a hindpaw. In control animals, pain-related behavioral reactions (licking and flinching) were higher in the evening (Dark) than in the morning (Light), both during the first (0–10 min) and the second (11–55 min) phase of the response to s.c. formalin. In animals pre-treated with luzindole, no diurnal changes occurred, pain reactions in the Dark being similar to those of the Light Control group. In a second group of experiments, artificial pinealectomy, obtained by exposing animals to continuous light for 48 h, also reduced pain reactions in the evening to levels comparable to those in the morning. Receptor autoradiography showed lower binding availability at spinal cord level in mice sacrificed during the Dark, as expected from the circadian pattern of melatonin secretion. A further significant decrease of melatonin receptor binding was induced by noxious stimulation. These results suggest a proalgesic role of endogenous melatonin in tonic pain.

Diurnal changes of tonic nociceptive responses in mice: evidence for a proalgesic role of melatonin

Diurnal variations in tonic pain reactions have been described in mice tested in Spring, but the underlying mechanisms are still unknown. We tested the potential role of melatonin, a key hormone in the control of neuro-endocrine circadian rhythms. The experiments were performed in male CBA/J mice housed under controlled temperature, humidity, and light (12/12 dark/light cycle) conditions, during the Light (7–10 a.m.) or Dark (7–10 p.m.) phases of the diurnal cycle. In a first group of experiments, animals were either pretreated with i.p. saline (controls) or with the melatonin receptor antagonist, luzindole (30 mg/kg), before the s.c. injection of a dilute formalin solution into a hindpaw. In control animals, pain-related behavioral reactions (licking and flinching) were higher in the evening (Dark) than in the morning (Light), both during the first (0–10 min) and the second (11–55 min) phase of the response to s.c. formalin. In animals pre-treated with luzindole, no diurnal changes occurred, pain reactions in the Dark being similar to those of the Light Control group. In a second group of experiments, artificial pinealectomy, obtained by exposing animals to continuous light for 48 h, also reduced pain reactions in the evening to levels comparable to those in the morning. Receptor autoradiography showed lower binding availability at spinal cord level in mice sacrificed during the Dark, as expected from the circadian pattern of melatonin secretion. A further significant decrease of melatonin receptor binding was induced by noxious stimulation. These results suggest a proalgesic role of endogenous melatonin in tonic pain.

Serotonin(7) receptors (5-HT(7)Rs) and their ligands.

Serotonin (5-HT) is involved in various physiological and pathological processes by interaction with 14 distinct receptor subtypes, grouped in seven classes of receptors (5-HT(1-7)) on the basis of amino acid sequence, pharmacology, and signal transduction pathways. The 5-HT(7)R is a G-protein coupled receptor with seven transmembrane domains. It was found by the application of molecular cloning and has been identified in rat, mouse, human, pig, and guinea pig. Although the biological functions of the 5-HT(7)Rs are poorly understood, preliminary evidence suggests that it may be involved in depression, control of circadian rhythms, and relaxation of vascular smooth muscles. For these reasons, the 5-HT(7)R has become a target for the development of novel drugs. This review will give a brief introduction of the pharmacology of 5-HT(7)R (molecular structure, distribution of 5-HT(7)R mRNA, localization of the 5-HT(7)R protein, functional correlates, and therapeutic potential) and a detailed survey of the 5-HT(7)R ligands, which have appeared in the literature in both papers and patents. Structure-activity relationships (SAR) of these ligands will also be described.

5-HT7 Receptors

Following the cloning of the 5-HT(7) receptor in 1993, studies to investigate 5-HT(7) receptor function in native tissues focused on identifying functional correlates that matched the pharmacological profile determined for the cloned receptor. Studies in peripheral tissues established that the 5-HT(7) receptor mediates the relaxation of smooth muscle, including the gastrointestinal tract and cardiovascular systems. Although a number of studies provided preliminary evidence for a role for the 5-HT(7) receptor in the circadian pacemaker function of the suprachiasmatic nucleus (SCN), additional studies to investigate 5-HT(7) receptor function in other brain regions have, until recently, been hindered by the absence of 5-HT(7) receptor-selective ligands. More recently, a number of 5-HT(7) receptor-selective antagonists including, SB-269970-A and SB-656104-A have been developed. Studies utilising these compounds suggest that the 5-HT(7) receptor modulates neuronal function in a number of brain areas including the hippocampus and thalamus. In turn, these findings suggest that 5-HT(7) receptor-selective ligands might prove therapeutically useful for the treatment of psychiatric disorders. In this respect there is increasing evidence to suggest that the 5-HT(7) receptor plays a role in the control of both circadian rhythms and sleep and might therefore represent a therapeutic target for the treatment of those disorders in which disturbances in circadian rhythms and sleep architecture are thought to be contributory factors. Furthermore, there is evidence to suggest that the receptor may play a role in other CNS disorders including, anxiety, cognitive disturbances and also migraine probably via both peripheral and central mechanisms. Although further studies are required to confirm the potential role of the receptor in such disorders, findings to date suggest there are exciting opportunities for the development of novel therapeutic agents acting either selectively at the 5-HT(7) receptor or whose profile of action includes an interaction with this receptor.

The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm.

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.

Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells

Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3′,5′-monophosphate (cAMP) in cultured retinal cells entrained to a daily light–dark (LD) cycle, as well as the role of Ca 2+ and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the noctural increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca 2+ influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca 2+ channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca 2+ influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

An essential role for peptidergic signalling in the control of circadian rhythms in the suprachiasmatic nuclei.

Two structurally related neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP), colocalized with glutamate in neurones of the retinohypothalamic tract, and vasoactive intestinal peptide (VIP), present in light-responsive cells of the suprachiasmatic nuclei (SCN), appear to play distinct and important roles in the control of mammalian circadian rhythms. Mice deficient in the PACAP-selective PAC1 receptor exhibit altered responsiveness of the SCN clock to light-induced phase-shifts, but display robust circadian patterns of wheel-running behaviour. By contrast, our studies of mice lacking the VPAC2 receptor, which responds to both PACAP and VIP, indicate that this receptor plays a critical role in rhythm generation in the SCN. The predominant factor determining wheel-running activity in VPAC2 receptor null (Vipr2-/-) mice is "masking" by light. Mutant animals re-entrain immediately to advances or delays in the light/dark cycle and do not exhibit robust circadian rhythms of behaviour when in constant darkness. The mice do not exhibit circadian expression of core clock genes (mPer1, mPer2, mCry1), or of the clock-controlled gene arginine vasopressin (AVP), in the SCN. We propose that VIP signalling between SCN neurones provides a paracrine reinforcing signal that is essential for sustained rhythm generation. The presence of VIP signalling in the SCN may explain why SCN neurones are capable of generating long-lasting self-sustained oscillations, whereas rhythmic clock gene expression in other tissues is dependent on periodic reinforcement by neural or hormonal signals.

Gas-Driven Transcriptional Regulation

Gene expression in mammalian cells is controlled by environmental and metabolic cues, but little is known about the molecular mechanisms that sense and respond to these cues. Dioum et al. (see the Perspective by Boehning and Snyder) have discovered one intriguing mechanism in a study of NPAS2 (neuronal PAS domain protein 2), a transcription factor implicated in the control of circadian rhythms. NPAS2 was shown to bind heme as a prosthetic group, and the heme, in turn, was shown to function as a gas-regulated sensor. In experiments with purified proteins, carbon monoxide (CO) was identified as a candidate ligand for this sensor. Exposure to CO inhibited the dimerization of NPAS2 with BMAL1, the protein that confers the transcription factor with DNA binding activity. E. M. Dioum, J. Rutter, J. R. Tuckerman, G. Gonzalez, M.-A. Gilles-Gonzalez, S. L. McKnight, NPAS2: A gas-responsive transcription factor. Science 298 , 2385-2387 (2002). [Abstract] [Full Text] D. Boehning, S. H. Snyder, Carbon monoxide and clocks. Science 298 , 2339-2340 (2002). [Summary] [Full Text]

Gas-Driven Transcriptional Regulation

Gene expression in mammalian cells is controlled by environmental and metabolic cues, but little is known about the molecular mechanisms that sense and respond to these cues. Dioum et al. (see the Perspective by Boehning and Snyder) have discovered one intriguing mechanism in a study of NPAS2 (neuronal PAS domain protein 2), a transcription factor implicated in the control of circadian rhythms. NPAS2 was shown to bind heme as a prosthetic group, and the heme, in turn, was shown to function as a gas-regulated sensor. In experiments with purified proteins, carbon monoxide (CO) was identified as a candidate ligand for this sensor. Exposure to CO inhibited the dimerization of NPAS2 with BMAL1, the protein that confers the transcription factor with DNA binding activity.E. M. Dioum, J. Rutter, J. R. Tuckerman, G. Gonzalez, M.-A. Gilles-Gonzalez, S. L. McKnight, NPAS2: A gas-responsive transcription factor. Science 298, 2385-2387 (2002). [Abstract] [Full Text]D. Boehning, S. H. Snyder, Carbon monoxide and clocks. Science 298, 2339-2340 (2002). [Summary] [Full Text]

Endogenous control of circadian rhythms of pheromone production in the turnip moth, Agrotis segetum.

The circadian variation of pheromone production in the turnip moth, Agrotis segetum, was characterized by quantifying (Z)-7-dodecenyl acetate (Z7-12:OAc), the most abundant pheromone component produced by female turnip moth, at different times of day. Under 17:7 h light-dark cycle (LD), the peak of Z7-12:OAc production occurred around 4 h into the scotophase, while there was very little pheromone production during the photophase. When females were maintained under constant darkness (DD), the periodicity of pheromone production was sustained for 3 consecutive days. Furthermore, the rhythm in pheromone production could be entrained to a shifted LD. These results demonstrate that the pheromone production in the turnip moth is regulated endogenously by a circadian clock. To understand how the circadian rhythm of pheromone production is generated, circadian variation of pheromone- biosynthesis-activating neuropeptide (PBAN)-like activity in the brain-suboesophageal ganglion complexes (Br-SOG), hemolymph, and ventral nerve cord (VNC) was also examined. Under both LD and DD, only the VNC displayed a circadian variation in the PBAN-like activity, which was significantly higher during the late-photophase than that in the scotophase. In addition, the present study showed that removal of VNC in isolated abdomen did not affect PBAN stimulation of pheromone production, while severing the VNC impaired normal pheromone production. The role of Br-SOG, VNC, and hemolymph in the regulation of the periodicity of pheromone production is discussed.

Pineal gland hormone and idiopathic scoliosis: possible effect of melatonin on sleep-related postural mechanisms.

Experimental and clinical evidences indicate that endocrine mechanisms, particularly involving the pineal gland, exert a role in the development of postural deficits leading to the occurrence of idiopatic scoliosis (IS). In particular, experiments performed in bipedal animals have shown that removal of the pineal gland, which secretes melatonin (M), induced a scoliosis, and that in such preparations, administration of this hormone prevented the development of this deformity (cf. 131). It appears also that adolescents with IS showed a reduced level of serum M with respect to age-related control subjects. The possible mechanisms involved in the M regulation of the tonic contraction of the axial musculature have been discussed. It is known that the pineal gland is implicated in the control of circadian rhythms, including the sleep-waking cycle, and that during this cycle there are prominent changes in postural activity, which affect not only the limbs, but also the axial musculature. These changes are characterized by a decrease followed by a suppression of postural activity, which occur particularly during transition from wakefulness to synchronized sleep and, more prominently, to rapid eye movement (REM) sleep. Episodes of postural atonia may also occur during the cataplectic episodes, which are typical of narcolepsy. Cholinergic and/or cholinoceptive neurons located in the dorsal pontine reticular formation (pRF) and the related medullary inhibitory reticulospinal (RS) system, intervene in the suppression of posture during REM sleep, as well as during the cataplectic episodes which occur in narcolepsy. These structures are under the modulatory (inhibitory) influence of the dorsomedial and the dorsolateral pontine tegmentum, where serotoninergic raphe nuclei (RN) neurons and noradrenergic locus coeruleus (LC) neurons are located. We postulated that M may act not only on the circadian pacemaker, but also directly on the pontine tegmental structures involved in the regulation of posture during the animal states indicated above. This hypothesis is supported by the facts that: 1) the dorsal pRF may contain specific binding sites for M; 2) this structure is particularly sensitive to M in adolescents, as well as in adult subjects affected by narcoleptic disturbances leading to cataplexy; 3) M increases the release of serotonin (5-HT), a neurotransmitter which enhances the postural tone by acting on the dorsal pRF: on the other hand, deficits in M levels may lower the activity of the serotoninergic raphe system, thus leading to a decrease or suppression of postural activity similar to that occurring either during REM sleep or during the cataplectic episodes typical of narcoleptic patients; 4) IS patients may show episodes of sleep apnea, a phenomenon which has been attibuted to a reduced tonic contraction of primary and accessory respiratory muscles during REM, resulting from a reduced release of 5-HT at dorsal pontine level. It has been postulated that, if the reduced M and 5-HT levels are subliminal to produce a complete suppression of posture under the conditions reported above, the reduced postural tone, which results from this condition may lead to the development of IS, due to hypotonia which affects the axial musculature. M secretion could be regulated not only by the activity of the serotoninergic raphe neurons projecting to the pineal gland, but probably also by the activity of noradrenergic LC neurons. It is likely that the development of IS, which results from a reduced level of M and 5-HT, may occur provided that the noradrenergic LC inhibition of the pontine structures is impaired. Such impairment could depend upon genetic factors, similar to those postulated to play a role in narcolepsy. In conclusion, the possibility exists that an impaired activity of brain monoaminergic systems may lead to disfunction in the production of M, which is apparently an important factor in the etiopathogenesis of IS.

The Eye Lens Protein αA-crystallin of the Blind Mole Rat Spalax ehrenbergi: Effects of Altered Functional Constraints

The rudimentary eyes of the mole rat Spalax ehrenbergi have lost their visual function, but are still required for the control of circadian rhythms. It has previously been found that αA-crystallin, a major eye lens protein in other mammals, evolved much faster in the mole rat than in rodents with normal vision. Yet, although mole rat αA-crystallin seems superfluous as a lens protein, its rate of change is still much slower than that of pseudogenes, suggesting some remaining function. The authors therefore studied the structure and function of recombinant mole rat αA-crystallin. Circular dichroism (CD), tryptophan fluorescence and gel permeation analyses indicated that the overall structure and stability of mole rat αA-crystallin are comparable to that of rat αA-crystallin. However, the chaperone-like activity of mole rat αA-crystallin is considerably lower than that of its rat orthologue. Two-dimensional NMR spectroscopy of mole rat αA-crystallin suggests that this may be in part due to a diminished flexibility of the C-terminal extension, which is thought to be important for the chaperoning capacity. Overall, mole rat αA-crystallin appears to still be a viable protein, confirming that it has some as yet elusive role, despite the loss of its primary lens function.

The APRR1/TOC1 quintet implicated in circadian rhythms of Arabidopsis thaliana: II. Characterization with CCA1-overexpressing plants.

We previously identified a novel class of proteins, named pseudo-response regulators (APRRs) in Arabidopsis thaliana, each of which (APRR1, APRR3, APRR5, APRR7, and APRR9) has an intriguing structural design containing an N-terminal pseudo receiver domain and a C-terminal CONSTANS motif. Among them, APRR1 is identical to TOC1, previously proposed to be a candidate component of an Arabidopsis circadian clock. Intriguingly, expressions of the APRR1/TOC1 family of genes are under control of coordinate circadian rhythms at the level of transcription, in the manner that each APRR-transcript starts accumulating sequentially after dawn with 2 to 3 h intervals in the order: APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1. Here we examined this circadian-related event, "circadian waves of the APRR1/TOC1 quintet", by employing CCA1-overexpression (CCA1-ox) transgenic plants, based on the fact that CCA1 is a well-characterized and the most plausible oscillator component. It was found that aberrant overexpression of the CCA1 gene severely perturbed free-running and sequential rhythms of the APRR1/TOC1 family of genes. In the accompanying paper, it was shown that overexpression of APRR1 also results in a marked alteration of the CCA1 circadian rhythm, and vice versa. Taken together, it was suggested that there are intimate and mutual links between these two types of circadian-associated components (APRRs and CCA1).