A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT K/R ) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT K/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT K/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.Circadian rhythms are molecular, physiological, or behavioral processes which occur with a periodic oscillation of approximately 24 h. These rhythms can be entrained by environmental cues such as light/dark cycles, but they persist in the absence of such cues, thus demonstrating the presence of an endogenous clock (reviewed in reference 40). Drosophila melanogaster has been widely used as a model organism for a genetic analysis of circadian rhythms, and many of the gene products comprising the endogenous clock mechanism were identified in this organism, including the first discovered clock gene, period (per) (reviewed in reference 42). Molecular genetic analysis of these rhythms has elucidated a basic clock mechanism consisting of oscillating clock gene products, which regulate their own expression through positive and negative feedback loops. During the night, PER and Timeless protein (TIM) levels rise in the cytoplasm, where the proteins heterodimerize, and then translocate into the nucleus to negatively regulate the transcription of their own genes and other genes. Negative regulation is effected by interactions with the transcription factors dClock (dCLK) and Cycle (CYC), which activate transcription of the per and tim genes and indirectly repress transcription of dClk in the absence of PER and TIM. Throughout the day, TIM levels do not accumulate in the nucleus because of TIM's degradation via a light-and cryptochrome-mediated degradation pathway (reviewed in reference 42).DBT, an ortholog of mammalian casein kinase Iε and casein kinase I␦ (CKIε/␦), regulates PER cytoplasmic and nuclear accumulation by triggering PER's degradation and regulating the timing of its nuclear accumulation (3,8,9,22,23,43,46,49). DBT's activity on PER is supplemented by the activity of CKII (2, 27, 28) and SGG (31) and is antagonized by a rhythmically expressed protein phosphatase (47). DBT may regulate other aspects of PER function (35) and ot...

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt S ) and long-period (dbt L ) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt S and dbt L mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT S , and DBT L proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt S and dbt L mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKI␦ expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt S and dbt L mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.Many daily biochemical, physiological, and behavioral processes are termed circadian rhythms because they are temporally regulated by an endogenous circadian clock. While these endogenous clocks are usually synchronized by the environmental light-dark or temperature cycle, in the absence of environmental cues their oscillations persist with a period of approximately 24 h (reviewed in reference 48). A genetic analysis in Drosophila melanogaster, as well as in other model organisms, has revealed much about the molecular components and mechanism of the circadian clock (reviewed in reference 74). Recently, it has become clear that the mammalian circadian clock mechanism is quite similar to the Drosophila mechanism (reviewed in reference 4).Central to the molecular mechanism of Drosophila are the oscillations of the per, tim, and dClk gene products, which drive transcriptional-translational feedback loops (3,5,16,24,27,37,59,60,75; reviewed in reference 72). PER and TIM proteins accumulate during the night, become phosphorylated, dimerize, and enter the nucleus (14,18,22,29,47,50,56,61,70,76,77), where they negatively regulate transcription of their own mRNAs (11,16,17,26,27,43,63,71,75) and positively regulate transcription of the dClk mRNA (5, 24). Both the negative and positive feedback regulation are thought to result from direct protein-protein interactions of PER and/or TIM with a CLK/CYC transcription factor (11, 37, 71) which, in the absence of PER and TIM, activates transcription of per, tim, and vrille (3,7,11,16,17,24,26,37,43,54,71) and represses the transcription of dClk (15,23,24). Entrainment, or synchronization of the clock to light-dark cycles, is conferred in part by a crypt...

Continuous disruption of circadian rhythms, as seen in human shift workers, has been associated with the development of a number of adverse mental and physiological conditions. However, scientific evidence linking circadian disruption to overall health, particularly in animal models, is not well documented. In this study, we have demonstrated that exposing C57BL/6J mice to 12-h phase shifts every 5 days for 3 mo had no effect on body weight or intestinal physiology. However, when animals were further challenged with dextran sodium sulfate to induce colitis, chronic shifting of the light-dark cycle led to a dramatic increase in the progression of the colitis as indicated by reduced body weight, abnormal intestinal histopathology, and an exacerbated inflammatory response. These data indicate that circadian disruption is an important predisposing factor that may provoke the onset or worsening of various disease states such as inflammatory disorders. This study provides further evidence for continued investigations using animal models of circadian disruption to examine the consequences of circadian disruption on health when organisms are faced with a "challenging" environment.

Background and aim We recently showed that patients with inflammatory bowel disease (IBD) report significantly more sleep disturbances. To determine whether disrupted sleep can affect the severity of inflammation and the course of IBD, we used an animal model of colonic inflammation to determine the effects of acute and chronic intermittent sleep deprivation on the severity of colonic inflammation and tissue damage in colitis and recovery from this damage. Methods Acute sleep deprivation (ASD) consisted of 24 h of forced locomotor activity in a mechanical wheel rotating at a constant speed. Chronic intermittent sleep deprivation (CISD) consisted of an acute sleep deprivation episode, followed by additional sleep deprivation periods in the wheel for 6 h every other day throughout the 10 day study period. To induce colitis, mice were given 2% dextran sodium sulfate (DSS) in their daily drinking water for 7 days. The development and severity of colitis were monitored by measuring weight loss and tissue myeloperoxidase (MPO) activity daily and colon histology scores 10 days after initiation of colitis. Results ASD or CISD did not cause colonic inflammation in vehicle-treated mice. Changes in daily body weight, tissue MPO levels and colon histopathology score were similar between mice that were sleep deprived and controls. Daily DSS ingestion caused colitis in mice. ASD worsened colonic inflammation: tissue MPO levels in ASD/DSS-treated mice were significantly higher than in DSS-treated mice that were not sleep deprived. However, the worsening of colonic inflammation by ASD was not enough to exacerbate clinical manifestations of colitis such as weight loss. In contrast, the deleterious effects of CISD were severe enough to cause worsening of histological and clinical manifestations of colitis. The deleterious effects of sleep deprivation on severity of colitis appeared to be due to both increased colonic inflammation and a decrease in the ability of mice to recover from DSS-induced colonic injury. Conclusion Both acute and chronic intermittent sleep deprivation exacerbate colonic inflammation. Thus, sleep deprivation could be an environmental trigger that predisposes IBD patients to develop flare ups and a more severe disease course. These results provide a scientific rationale to conduct an interventional trial to determine whether improvement in sleep patterns will prevent IBD flare ups, modify the disease course, and improve quality of life.

Despite decades of research in defining sleep-wake properties in mammals, little is known about the nature or identity of genes that regulate sleep, a fundamental behaviour that in humans occupies about one-third of the entire lifespan. While genome-wide association studies in humans and quantitative trait loci (QTL) analyses in mice have identified candidate genes for an increasing number of complex traits and genetic diseases, the resources and time-consuming process necessary for obtaining detailed quantitative data have made sleep seemingly intractable to similar large-scale genomic approaches. Here we describe analysis of 20 sleep-wake traits from 269 mice from a genetically segregating population that reveals 52 significant QTL representing a minimum of 20 genomic loci. While many (28) QTL affected a particular sleep-wake trait (e.g., amount of wake) across the full 24-hr day, other loci only affected a trait in the light or dark period while some loci had opposite effects on the trait during the light vs. dark. Analysis of a dataset for multiple sleep-wake traits led to previously undetected interactions (including the differential genetic control of number and duration of REM bouts), as well as possible shared genetic regulatory mechanisms for seemingly different unrelated sleep-wake traits (e.g., number of arousals and REM latency). Construction of a Bayesian network for sleep-wake traits and loci led to the identification of sub-networks of linkage not detectable in smaller data sets or limited single-trait analyses. For example, the network analyses revealed a novel chain of causal relationships between the chromosome 17@29cM QTL, total amount of wake, and duration of wake bouts in both light and dark periods that implies a mechanism whereby overall sleep need, mediated by this locus, in turn determines the length of each wake bout. Taken together, the present results reveal a complex genetic landscape underlying multiple sleep-wake traits and emphasize the need for a systems biology approach for elucidating the full extent of the genetic regulatory mechanisms of this complex and universal behavior.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Ie/d (CKIe/d) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI shortperiod mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIe/d. Moreover, expression of either Drosophila DBT or the vertebrate CKId kinase carrying the Drosophila dbt S or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKId carrying the dbt L mutation does not lengthen circadian rhythms, while Drosophila DBT L does. Different effects of the dbt S and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKId and of the functions affected by their period-shortening mutations.

Stroke is the leading cause of serious, long-term disability in the United States. Impairment of upper extremity function is a common outcome following stroke, often to the detriment of lifestyle and employment opportunities. While the upper extremity is a natural target for therapy, treatment may be hampered by limitations in baseline capability as lack of success may discourage arm and hand use. We developeda virtual reality (VR) system in order to encourage repetitive task practice. This system combined an assistive glove with a novel VR environment. A set of exercises for this system was developed to encourage specific movements. Six stroke survivors with chronic upper extremity hemiparesis volunteered to participate in a pilot study in which they completed 18 one-hour training sessions with the VR system. Performance with the system was recorded across the 18 training sessions. Clinical evaluations of motor control were conducted at three time points: prior to initiation of training, following the end of training, and 1 month later. Subjects displayed significant improvement on performance of the virtual tasks over the course of the training, although for the clinical outcome measures only lateral pinch showed significant improvement. Future expansion to multi-user virtual environments may extend the benefits of this system for stroke survivors with hemiparesis by furthering engagement in the rehabilitation exercises.

No abstract