A role of cryptochrome for magnetic field-dependent improvement of sleep quality, lifespan, and motor function in Drosophila.

Understanding the molecular genetic basis of animal magnet reception has been one of the big challenges in molecular biology. Recently it was discovered that the magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here, using the fruit fly as a magnet-receptive model organism, we show that the magnetic field exposure (0.4-0.6 mT) extended lifespan under starvation, but not in cryptochrome mutant flies (cryb ). The magnetic field exposure increases motor function in wild type and neurodegenerative disease model flies. Furthermore, the magnetic field exposure improved sleep quality at night-time specific manner, but not in cryb . We also showed that repeated AC magnetic field exposure increased climbing activity in wild-type Drosophila, but not in cryb . The data suggests that magnetic field-dependent improvement of lifespan, sleep quality, and motor function is mediated through a cry-dependent pathway in Drosophila.

Novel hypnotics of Japanese traditional herbal medicines to caffeine-induced insomnia in Drosophila by using Newly-developed automated sleep and rhythm analysis system (AutoCircaS).

Sleep in Drosophila was defined in the year 2000 by using Drosophila Activity Monitor (DAM) system. But DAM is very small tube space and one fly per tube is very limited to analyze for fly social behavior. To overcome such demerits of DAM system, we developed a novel automated sleep and rhythm analysis system (AutoCircaS) which monitors and records any behaviors like social mating, sleep, and circadian rhythm in flies (Drosophila) and small fishes medaka (Oryzias latipes) in free space using the time-lapse (one frame per 10 sec) imaging. AutoCircaS can detect the caffeine-induced insomnia in flies in light-dark (LD) and constant dark (DD) conditions. Thus, using the AutoCircaS, we discovered that Japanese traditional herbal medicines, KyushinKannouGan-ki (KKG), NouKassei (NK) as well as, and Sansoninto, significantly improved caffeine-induced insomnia in flies. The data suggest that AutoCircaS is useful for sleep analysis of small animals and screening of new sedative-hypnotics from many origins.

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition.

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.

Saffron ameliorated motor symptoms, short life span and retinal degeneration in Parkinson's disease fly models.

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linné (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

Effects of an electric field on sleep quality and life span mediated by ultraviolet (UV)-A/blue light photoreceptor CRYPTOCHROME in Drosophila.

Although electric fields (EF) exert beneficial effects on animal wound healing, differentiation, cancers and rheumatoid arthritis, the molecular mechanisms of these effects have remained unclear about a half century. Therefore, we aimed to elucidate the molecular mechanisms underlying EF effects in Drosophila melanogaster as a genetic animal model. Here we show that the sleep quality of wild type (WT) flies was improved by exposure to a 50-Hz (35 kV/m) constant electric field during the day time, but not during the night time. The effect was undetectable in cryptochrome mutant (cryb) flies. Exposure to a 50-Hz electric field under low nutrient conditions elongated the lifespan of male and female WT flies by ~ 18%, but not of several cry mutants and cry RNAi strains. Metabolome analysis indicated that the adenosine triphosphate (ATP) content was higher in intact WT than cry gene mutant strains exposed to an electric field. A putative magnetoreceptor protein and UV-A/blue light photoreceptor, CRYPTOCHROME (CRY) is involved in electric field (EF) receptors in animals. The present findings constitute hitherto unknown genetic evidence of a CRY-based system that is electric field sensitive in animals.

Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials.

Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion. Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study.

Epidermal growth factor (EGF) treatment on multipotential stromal cells (MSCs). Possible enhancement of therapeutic potential of MSC.

Adult bone marrow multipotential stromal cells (MSCs) hold great promise in regenerative medicine and tissue engineering. However, due to their low numbers upon harvesting, MSCs need to be expanded in vitro without biasing future differentiation for optimal utility. In this concept paper, we focus on the potential use of epidermal growth factor (EGF), prototypal growth factor for enhancing the harvesting and/or differentiation of MSCs. Soluble EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population, and thus did not induce differentiation. However, tethered form of EGF was shown to promote osteogenic differentiation. Soluble EGF was also shown to increase paracrine secretions including VEGF and HGF from MSC. Thus, soluble EGF can be used not only to expand MSC in vitro, but also to enhance paracrine secretion through drug-releasing MSC-encapsulated scaffolds in vivo. Tethered EGF can also be utilized to direct MSC towards osteogenic lineage both in vitro and in vivo.

Effects of Kamikihito and Unkei-to on Sleep Behavior of Wild Type and Parkinson Model in Drosophila.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and it is associated with sleep behavior disorders. In Drosophila melanogaster disease model, human α-synuclein A30P overexpressing flies (A30P PD model) have been shown for levy body aggregation and movement disorders. We measured sleep rhythms in the A30P PD model flies using the Drosophila Activity Monitoring system and found that they develop sleep defects at 20 days after eclosion. Furthermore, the total amount of sleep is significantly reduced in middle-aged PD model flies and the reduction has been attributed to nighttime sleep. The number and length of sleep bouts also decreased in middle-aged A30P PD model flies. Feeding of the oriental traditional herbal medicines (Kampo), Kamikihito and Unkei-to significantly ameliorate the level of sleep defects in A30P PD model flies. The Kamikihito and Unkei-to recovered 60-min sleep bouts number in the A30P PD model flies to the level of young (5 days after eclosion) flies. Kamikihito recovered sleep both in wild-type and PD model flies. Unkei-to ameliorates not only sleep but also motor function in PD model flies. The data suggest that Kamikihito and Unkei-to might be useful for the sleep defects in human PD patients as well as healthy human.

Minos-insertion mutant of the Drosophila GBA gene homologue showed abnormal phenotypes of climbing ability, sleep and life span with accumulation of hydroxy-glucocerebroside.

Gaucher's disease in humans is considered a deficiency of glucocerebrosidase (GlcCerase) that result in the accumulation of its substrate, glucocerebroside (GlcCer). Although mouse models of Gaucher's disease have been reported from several laboratories, these models are limited due to the perinatal lethality of GlcCerase gene. Here, we examined phenotypes of Drosophila melanogaster homologues genes of the human Gaucher's disease gene by using Minos insertion. One of two Minos insertion mutants to unknown function gene (CG31414) accumulates the hydroxy-GlcCer in whole body of Drosophila melanogaster. This mutant showed abnormal phenotypes of climbing ability and sleep, and short lifespan. These abnormal phenotypes are very similar to that of Gaucher's disease in human. In contrast, another Minos insertion mutant (CG31148) and its RNAi line did not show such severe phenotype as observed in CG31414 gene mutation. The data suggests that Drosophila CG31414 gene mutation might be useful for unraveling the molecular mechanism of Gaucher's disease.

Inositols affect the mating circadian rhythm of Drosophila melanogaster.

Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. Here, we showed that the close-proximity (CP) rhythm of D. melanogaster courtship behavior was damped under low-nutrient conditions, but significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, we found that myo-inositol increased the amplitude and slightly shortened the period of the CP rhythm. Real-time reporter assays showed that myo-inositol and D-pinitol shortened the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals.

The circadian binding of CLOCK protein to the promoter of C/ebpα gene in mouse cells.

C/EBPα plays important roles in metabolism as well as in the maintenance of energy homeostasis. Here we describe loss of the circadian oscillation of C/ebpα expression in liver of Clock mutant mice. Reporter assays indicate Clock and Bmal significantly induced C/ebpα gene expression whereas Cry suppressed. Real time reporter assays showed that two mutated E-boxes disrupted C/ebpα promoter dependent-oscillation. Chromatin immunoprecipitation suggests Clock can bind to two E-boxes in the C/ebpα promoter with a circadian manner in vivo. Thus, C/ebpα gene transcription is under circadian control of a core clock component, Clock. The data suggests that circadian disturbances may affect metabolic abnormalities through the C/ebpα pathway in liver.

Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages.

BACKGROUND: Periodontitis is prevalent in older humans. Limiting the inflammation associated with periodontitis may provide a therapy for this condition, because Gram-negative bacteria expressing lipopolysaccharide (LPS) have a key role in initiation of inflammation by activating macrophage functions. Because oxidized galectin-1 regulates macrophage functions in other systems, we sought to establish whether this galectin-1 mRNA is expressed in the oral cavity, and whether it could dampen LPS-induced macrophage activation in vitro. METHODS: Using the reverse transcriptase polymerase chain reaction (RT-PCR), we measured galectin-1 mRNA expression to clarify its localization to rat gingival tissues and studied the effect of Porphyromonas gingivalis challenge on galectin-1 expression. Next, we tested the effects of adding oxidized galectin-1 to cultured LPS-activated peritoneal macrophages on mRNA expression of proinflammatory factors by RT-PCR and real-time RT-PCR. RESULTS: We established that galectin-1 mRNA is expressed in gingival tissues and also showed that galectin-1 mRNA was significantly increased by challenge with P. gingivalis, indicating that galectin-1 may regulate oral inflammation. On the other hand, LPS 100 ng/mL in serum-containing medium induced macrophages to upregulate mRNA associated with a proinflammatory response, ie, interleukins 1ß and 6, and inducible nitric oxide synthase. We showed that application of 0.1-10 ng/mL of oxidized galectin-1 to LPS-treated macrophages reduced the intense LPS- induced increase by serum in proinflammatory mRNA expression in a concentration-dependent manner. Furthermore, application of oxidized galectin-1 10 ng/mL to LPS-treated macrophages in serum-free medium also showed a similar effect on LPS activity. CONCLUSION: Oxidized galectin-1 restricts the proinflammatory actions of LPS, and this protein could limit the negative effects of inflammation.

Development of virtual reality exercise of hand motion assist robot for rehabilitation therapy by patient self-motion control.

This paper presents a virtual reality-enhanced hand rehabilitation support system with a symmetric master-slave motion assistant for independent rehabilitation therapies. Our aim is to provide fine motion exercise for a hand and fingers, which allows the impaired hand of a patient to be driven by his or her healthy hand on the opposite side. Since most disabilities caused by cerebral vascular accidents or bone fractures are hemiplegic, we adopted a symmetric master-slave motion assistant system in which the impaired hand is driven by the healthy hand on the opposite side. A VR environment displaying an effective exercise was created in consideration of system's characteristic. To verify the effectiveness of this system, a clinical test was executed by applying to six patients.

Regularity in an environment produces an internal torque pattern for biped balance control.

In this paper, we present a control method for achieving biped static balance under unknown periodic external forces whose periods are only known. In order to maintain static balance adaptively in an uncertain environment, it is essential to have information on the ground reaction forces. However, when the biped is exposed to a steady environment that provides an external force periodically, uncertain factors on the regularity with respect to a steady environment are gradually clarified using learning process, and finally a torque pattern for balancing motion is acquired. Consequently, static balance is maintained without feedback from ground reaction forces and achieved in a feedforward manner.

A Simple and Quick Method to Isolate Nuclear Extracts from Pupae of Drosophila melanogaster.

Preparation of nuclear extracts is a critical step for biochemical identification of factors which function in the nuclei such as transcription factors. We have established a new method to prepare nuclear extracts from prepupae or pupae of Drosophila melanogaster. The method is simple and particularly useful for small-scale isolation from materials that are hard to get in large amounts such as specific tissues or animal bodies covered by pupal cases.

BetaFTZ-F1 dependent and independent activation of Edg78E, a pupal cuticle gene, during the early metamorphic period in Drosophila melanogaster.

Insect metamorphosis is a developmentally important event for formation of adult structures from larval imaginal cells, and it is controlled by the ecdysteroid hormone. At the onset of metamorphosis, both the cuticle gene Edg78E and the transcription factor betaFTZ-F1 are expressed during the mid- to late prepupal period after a large ecdysteroid pulse. Edg78E mRNA is inducible by premature expression of betaFTZ-F1 and the Edg78E expression level is reduced in an ftz-f1 mutant. Using a transgenic fly reporter assay, a 1.2 kb promoter region of the Edg78E gene has been identified, which was sufficient for appropriate temporally and spatially specific expression of the reporter gene LacZ. Within the promoter region, two betaFTZ-F1 binding sites are present and disruption of these sites reduced the expression level of the reporter gene. LacZ expression levels were dramatically reduced in the head and thorax regions but not affected in the abdominal region, suggesting that betaFTZ-F1 is required for high-level Edg78E expression specifically in the head and thorax regions. The findings suggest that betaFTZ-F1 is a regulator for temporal gene expression at the onset of metamorphosis, and that complex mechanisms regulate the temporal and spatial regulation of gene expression during metamorphosis.