Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress.

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA(Met) (iMet) through 5'-3' exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells.

Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells.

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNA(Met) (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability.

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.

Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells.

Whole transcriptome analyses have revealed a large number of novel transcripts including long and short noncoding RNAs (ncRNAs). Currently, there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to cellular processes. In particular, nuclear long ncRNAs may be involved in controlling various aspects of biological regulation, such as stress responses. By a combination of bioinformatic and experimental approaches, we identified 25 novel nuclear long ncRNAs from 6,088,565 full-length human cDNA sequences. Some nuclear long ncRNAs were conserved among vertebrates, whereas others were found only among primates. Expression profiling of the nuclear long ncRNAs in human tissues revealed that most were expressed ubiquitously. A subset of the identified nuclear long ncRNAs was induced by the genotoxic agents mitomycin C or doxorubicin, in HeLa Tet-off cells. There were no commonly altered nuclear long ncRNAs between mitomycin C- and doxorubicin-treated cells. These results suggest that distinct sets of nuclear long ncRNAs play roles in cellular defense mechanisms against specific genotoxic agents, and that particular long ncRNAs have the potential to be surrogate indicators of a specific cell stress.

Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals.

Mammalian genomes produce huge numbers of noncoding RNAs (ncRNAs). However, the functions of most ncRNAs are unclear, and novel techniques that can distinguish functional ncRNAs are needed. Studies of mRNAs have revealed that the half-life of each mRNA is closely related to its physiological function, raising the possibility that the RNA stability of an ncRNA reflects its function. In this study, we first determined the half-lives of 11,052 mRNAs and 1418 ncRNAs in HeLa Tet-off (TO) cells by developing a novel genome-wide method, which we named 5'-bromo-uridine immunoprecipitation chase-deep sequencing analysis (BRIC-seq). This method involved pulse-labeling endogenous RNAs with 5'-bromo-uridine and measuring the ongoing decrease in RNA levels over time using multifaceted deep sequencing. By analyzing the relationship between RNA half-lives and functional categories, we found that RNAs with a long half-life (t(1/2) ≥ 4 h) contained a significant proportion of ncRNAs, as well as mRNAs involved in housekeeping functions, whereas RNAs with a short half-life (t(1/2) < 4 h) included known regulatory ncRNAs and regulatory mRNAs. The stabilities of a significant set of short-lived ncRNAs are regulated by external stimuli, such as retinoic acid treatment. In particular, we identified and characterized several novel long ncRNAs involved in cell proliferation from the group of short-lived ncRNAs. We designated this novel class of ncRNAs with a short half-life as Short-Lived noncoding Transcripts (SLiTs). We propose that the strategy of monitoring RNA half-life will provide a powerful tool for investigating hitherto functionally uncharacterized regulatory RNAs.

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles.

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

Formation of tRNA granules in the nucleus of heat-induced human cells.

The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes.

MALAT-1, a long non-coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short-interfering RNA-mediated MALAT-1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT-1 knockdown cells, pre-mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT-1 is a novel class of non-coding RNA that promotes cell motility through transcriptional and post-transcriptional regulation of motility related gene expression.

Nitrate toxicity on visceral organs of Medaka fish, Oryzias latipes: aiming to raise fish from egg to egg in space.

Histological survey was made to determine nitrate toxicity on the Medaka fish, Oryzias latipes. In order to investigate the effects of short-term exposure to nitrate, one-month-old Medaka fish was exposed to NaNO3 at concentrations of 100 and 125 mg NO3-N l-1 for 96 hours. At the end of the exposure period, survival rate was found to be 30% and 10%, for the 100 and 125 mg NO3-N l-1 exposure concentrations, respectively. Histological examination of the organs showed that disruption of cell alignment was a common feature in the gills, intestinal ampulla, liver and kidney. A long-term exposure experiment was also carried out, whereby Medaka fish was exposed to NaNO3 (100 and 125 mg NO3-N l-1) for three months from its egg stage. Eggs treated with NaNO3 hatched within 10 days after fertilization. At the end of the exposure period, survival rate in the 100 and 125 mg NO3-N l-1 treatments were 40% and 30%, respectively. Fibrosis of the hepatic cells and curved spinal column were observed in the juveniles subjected to long-term nitrate exposure. The results of our experiments suggest that the high mortality resulting from short-term acute exposure to nitrate is caused by general dysfunction throughout the whole body. The chronic toxic effects attributed to nitrate, following long-term exposure, were likely to have resulted from nutrient deficiency caused by hepatic dysfunction.

Ten years after medaka fish mated and laid eggs in space and further preparation for the life-cycle experiment on ISS.

In the second International Microgravity Laboratory (IML-2/STS-65) mission in 1994, medaka fish (Oryzias latipes) performed their successful mating behavior in space for the first time among vertebrate animals. The eggs the fish laid in space developed normally, and hatched as fry (baby fish) in space. Those fry born in space had the same number of germ cells as the ground control fish, and these germ cells later developed to produce the offspring on the ground. Fry hatched in space did not exhibit any looping behavior regardless of their strain, visual acuity, etc. For fish, one of the most exciting experiments to be done abroad the International Space Station (ISS) is a realization of fish life cycles in microgravity. At present, fish are the most likely candidates to be the first vertebrate to live their life cycle in space. Research done in our laboratory for realizing the life-cycle experiment of medaka fish are also introduced.

Morphological characteristics of the kidney and lung in the neonatal rats observed after 16 days spaceflight.

The aim of this study is to examine the structural development in kidney and lung macroscopically which relate with cardiovascular system in rats raised in space. Twenty three nine-day old rats and six fifteen-day old rats, which were launched at these ages and nursed by their dams in the Space Shuttle Colombia for 16 days (STS-90; Neurolab). Seventeen animals of the nine-day old rats were defined as the nine-day group, and the rest was defined as the re-adaptation group, which were reared on the ground for 30 more days after landing. The organs were weighed and the ratio of the organ weight to the body weight (body weight ratio) was calculated. Both of lung and kidney in flight rats were significantly heavier than ground controls in the body weight ratio. We found that the kidney in the nine-day and the fifteen-day group tended to extend of dorsal-ventral length in macroscopic observations. However, this difference was not observed in the re-adaptation group. These results suggest that space environment may affect in kidney development. On the other hand, the lung had no differences in macroscopic structure among flight and control groups.

Behavior of Medaka fish under distributed gravity.

The threshold value of gravity for Medaka fish (Oryzias latipes) was determined using parabolic flights of an airplane. Rotating a turntable during a 20 sec of microgravity, a gradient field of centrifugal force was realized in the aquarium. Fish of HO5 strain were used because from the previous studies, in microgravity they were known to exhibit looping behavior more easily than any other strains. Looping fish became stable (i.e., recovered their posture control) when fish swam from a lower-gravity area of the aquarium to an area of a certain gravity value or beyond. On the other hand, stable fish lost their posture control and started looping when fish swam into an area of a gravity lower than a certain value. Using these phenomena, we obtained the gravity value of 0.21 to 0.26 G as for the threshold value for Medaka fish to sense the gravity.

Studies on vestibular organs in the fry of mutant Medaka.

We provide an overview of the features of vestibular organs, especially of otolith organs in mutant Medaka ha. The ha fish have a dysfunction in sensing gravity due to the absence of utricular otoliths either unilaterally or bilaterally. Especially in their early developmental stages, the posture control is predominantly depend on light rather than on gravity seen in the wild-type. The ha juveniles that were kept and tolerated in an environment with altered light directions indicated a strong light dependency on the posture maintenance, while the fish under an ordinary light environment gradually changed their posture control by sensing light to gravity around 30 days after hatching and beyond. Their behavior under microgravity agreed with such phenomena observed in ground laboratory.

Life-cycle experiments of medaka fish aboard the international space station.

Fish are the most likely candidates to be the first vertebrate to live their life cycle aboard the International Space Station (ISS). In the space-shuttle experiment using medaka, the fry born in space had the same number of germ cells as the ground control fish, and these germ cells later developed to produce the offspring on the ground. Fry hatched in space did not exhibit any looping behavior regardless of their strain, visual acuity, etc. The aquatic habitat (AQH) is a space habitat designed for long-term breeding of medaka, zebrafish and Xenopus, and recent advancements in this hardware also support fish life-cycle experiments. From the crosses between two strains, fish having good eyesight and less sensitivity to gravity were obtained, and their tolerance to microgravity was tested by parabolic flight using an airplane. The fish exhibited less looping and no differences in degree of looping between light and dark conditions. These are possible candidates for the first adult medaka (parent fish) to start a life cycle aboard ISS. Embryos were treated with a three-dimensional clinostat. Such simulated microgravity caused no differences in tissue architecture or in gene expression within the retina, nor in formation of cartilage (head skeleton). Otolith formation in embryos and fry was investigated for wild-type and mutant (ha) medaka. The ha embryos could not form utricular otoliths. They formed saccular otoliths but with a delay. Fry of the mutant fish lacking the utricular otoliths are highly light-dependent at the time of hatching, showing a perfect dorsal-light response (DLR). As they grow, they eventually shift from being light dependent to gravity dependent. Continuous treatment of the fry with altered light direction suppressed this shift to gravity dependence. Being less dependent on gravity, these fish can serve as model fish in studying the differences expected for the fish that have experienced a life cycle in microgravity.

[An easy introduction to organismal and cellular mechanisms for graviperception].

In this review paper, organismal and also cellular mechanisms for perception of gravity are explained. A statolith and a number of hair cells which surround the statolith is a basic structure of statocysts for detecting the direction of gravity or tilting of the body in various animals. The vestibular system of vertebrate was explained, especially on the process from the body-tilting to impulse frequencies which travel to brain. For the cellular responses to gravity, contribution of various organella (??) and cytoskeleton are introduced. Such cellular responses may change when the gravity values become less or null. Gravity perception mechanisms of plants are also explained.

[Effects of the spaceflight on organ-development in the neonatal rats: results in the Neurolab (STS-90)].

In the Neurolab mission, we found that spaceflight affects the development of the aortic baroreflex system and the body weight of the flight rats was significantly lighter [correction of lightess] than that of the control group. The aim of this study is to examine the structural and functional development in various tissues and organs. One hundred and eighteen nine-day old rats and seven fifteen-day old rats, which were launched at these ages and nursed by their dams in the space shuttle Columbia for 16 days, were served for this study. Two hundred and twenty one neonates were used as the ground controls (VIV: vivarium and AGC: asynchronous ground controls). On the landing day after they returned to the earth, the rats were perfused with a fixative under deep urethane anesthesia, and the organs were weighed and the ratio of the organ weight to the body weight was calculated. Six animals of the nine-day old group were reared on the ground for 30 more days after landing and also examined in the same protocol as the landing-day-examination. The organs obtained to examine were heart, lung, spleen, thymus, adrenal glands, kidney, liver, small intestine, large intestine, mesentery, pancreas, testis and ovary. Paraffin sections were made from some organ tissues and prepared for HE staining and immunohistochemistry. We compared these organs in the flight rat with those in the ground controls. All organs except the lung of nine-day old group were significantly smaller. In the ratio of organ weight to body weight, the lung and heart were significantly larger. The weight and ratio of the liver showed no significant difference. The thymus, spleen, mesentery and pancreas were smaller in the weight and the ratio. There were no differences in the body weight among 30-day reared groups, but the lung in the flight group is significantly heavier than the control groups and thymus also tends to be relatively heavy. In flight rats of the fifteen-day group, the kidney was heavy and the ovary was light as compared to the controls. The adipose tissue was macroscopically little found around the thoracic and abdominal organs in all rats of the flight group. These results suggest that the organs related to oxygen supply like as the lung and heart have priority in development over the mesentery and immune system organs even during spaceflight. Lightness of the mesentery in space rats is due to small contents of adipose tissues, and may reflect amounts of the food taken by the flight dams. Lightness of the organs like as the thymus, spleen and pancreas suggests that spaceflight may affect the immune system and also affect continuously the lung and thymus development even after landing.