東端 晃

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798–4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetyl-cysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcrip-tional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at-110 to-60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechano-transducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.

We investigated the effects of microgravity environment on growth and plant hormone levels in dark-grown rice shoots cultivated in artificial 1 g and microgravity conditions on the International Space Station (ISS). Growth of microgravity-grown shoots was comparable to that of 1 g-grown shoots. Endogenous levels of indole-3-acetic acid (IAA) in shoots remained constant, while those of abscisic acid (ABA), jasmonic acid (JA), cytokinins (CKs) and gibberellins (GAs) decreased during the cultivation period under both conditions. The levels of auxin, ABA, JA, CKs and GAs in rice shoots grown under microgravity conditions were comparable to those under 1 g conditions. These results suggest microgravity environment in space had minimal impact on levels of these plant hormones in rice shoots, which may be the cause of the persistence of normal growth of shoots under microgravity conditions. Concerning ethylene, the expression level of a gene for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in ethylene biosynthesis, was reduced under microgravity conditions, suggesting that microgravity may affect the ethylene production. Therefore, ethylene production may be responsive to alterations of the gravitational force.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (lG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under lG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and lG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in lG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α. Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

The effects of long-term exposure to extreme space conditions on astronauts were investigated by analyzing hair samples from ten astronauts who had spent six months on the International Space Station (ISS). Two samples were collected before, during and after their stays in the ISS; hereafter, referred to as Preflight, Inflight and Postflight, respectively. The ratios of mitochondrial (mt) to nuclear (n) DNA and mtRNA to nRNA were analyzed via quantitative PCR. The combined data of Preflight, Inflight and Postflight show a significant reduction in the mtDNA/nDNA in Inflight, and significant reductions in the mtRNA/nRNA ratios in both the Inflight and Postflight samples. The mtRNA/mtDNA ratios were relatively constant, except in the Postflight samples. Using the same samples, the expression of redox and signal transduction related genes, MnSOD, CuZnSOD, Nrf2, Keap1, GPx4 and Catalase was also examined. The results of the combined data from Preflight, Inflight and Postflight show a significant decrease in the expression of all of the redox-related genes in the samples collected Postflight, with the exception of Catalase, which show no change. This decreased expression may contribute to increased oxidative stress Inflight resulting in the mitochondrial damage that is apparent Postflight.

Reorientation of cucumber seedlings induces re-localization of CsPIN1 auxin efflux carriers in endodermal cells of the transition zone between hypocotyl and roots. This study examined whether the re-localization of CsPIN1 was due to the graviresponse. Immunohistochemical analysis indicated that, when cucumber seedlings were grown entirely under microgravity conditions in space, CsPIN1 in endodermal cells was mainly localized to the cell side parallel to the minor axis of the elliptic cross-section of the transition zone. However, when cucumber seeds were germinated in microgravity for 24 h and then exposed to 1g centrifugation in a direction crosswise to the seedling axis for 2 h in space, CsPIN1 was re-localized to the bottom of endodermal cells of the transition zone. These results reveal that the localization of CsPIN1 in endodermal cells changes in response to gravity. Furthermore, our results suggest that the endodermal cell layer becomes a canal by which auxin is laterally transported from the upper to the lower flank in response to gravity. The graviresponse-regulated re-localization of CsPIN1 could be responsible for the decrease in auxin level, and thus for the suppression of peg formation, on the upper side of the transition zone in horizontally placed seedlings of cucumber.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 angstrom. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy. (C) 2016 Elsevier Inc. All rights reserved.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P <= 0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.

Vibrio vulnificus (V. vulnificus)は，世界中の沿岸海水中に広く生息し，本菌に汚染された魚介類の生食や創傷からの菌の侵入により感染する．発熱，四肢の壊死性筋膜炎等の症状を呈し，短期間で死に至る極めて予後不良の疾患である．V. vulnificus感染症患者の多くは基礎疾患に肝硬変や肝癌等の肝機能障害を有する．日和見感染症である本症において有効なワクチンの開発が重要であり，免疫原性の高い抗原の同定が必要である．今回我々は，V. vulnificusのヒト血清における免疫原性の高い抗原を確認することを目的とし，V. vulnificus感染症患者10名の血清を用いた菌溶解液(V. vulnificus7株，V. parahaemolyticus1株)のimmunoblottingを施行し，感染による抗体産生を分析した．発症時からすでに見られる62-kDaのbandは，患者のみならずV. vulnificus感染症未発症の慢性肝機能障害者及び肝機能正常者のいずれの血清を用いたimmunoblottingにおいても共通して認められた．また，Escherichia.coliやKlebsiella pneumoniaeを用いたimmunoblottingにおいても同様の反応を認め，交差反応を起こしている可能性も考えられた．今回の実験から，V. vulnificusの62-kDaの蛋白がヒトにおける免疫原性の高い抗原の一つであることが予想された．Vibrio vulnificus infection can cause necrotizing fasciitis and sepsis and can develop within a few days despite intensive care. The mortality rate is up to 60% in vulnerable people. Most patients infected with this microbe have chronic liver disease, especially liver cirrhosis or cancer, as an underlying disease. V. vulnificus infection is opportunistic, and there is an urgent need to develop an anti-V. vulnificus vaccine. Thus, it is important to identify immunogenic antigens. We collected human sera from three subject groups : patients with V. vulnificus infection, patients with chronic liver disease but without V. vulnificus infection, and healthy volunteers with normal liver function. Immunoblots of cytosolic and membrane proteins of seven strains of V. vulnificus and one of V. parahaemolyticus were performed with sera from these groups. Although we could not demonstrate differences in antibody response between the groups, all sera showed a strong antibody response to a 62-kDa protein that was common to all strains examined. Immunoblots of Escherichia coli and Klebsiella pneumoniae also showed strong antibody response to this 62-kDa protein, and the possibility of cross-reaction cannot be denied. We identified this 62-kDa protein as an immunogenic antigen of V. vulnificus for humans.

Many astronauts stay in the International Space Station (ISS) for a long period of time. Therefore, the development of astronaut health care technologies is very important. Especially, an understanding of the effects of the space environment, such as microgravity and radiation, on protein, gene, and mineral metabolism is important for developing countermeasures against the adverse effects experienced by astronauts who are in space for long periods of time. Since December 2009, the Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples from ISS crew members who have been in space (experiment nicknamed "HAIR"). As animal control experiments, we could have an opportunity to analyze rodents samples by participating the tissue sharing program of space-flown mice organized by Italian Space Agency (AGI) and National Aeronautics and Space Administration (NASA). It will reasonably complement human hair experiment because we able to conduct more detailed skin analysis which is enable in human experiment. The purpose of this flown-mice experiment is to study the effects of long-term exposure to space environment. In this experiment, we analyzed mice skin contained hair roots. The samples were taken from space-flown (3-month and 2-week) and 3-month hindlimb suspensioned and 3-month 2G exposed mice, and ground-control mice. For the skin contained hair roots, the extracted and amplified RNA was used to DNA microarray analysis, and was further analyzed with expression on the interesting genes by real time Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. And the extracted protein was used to Mass Spectrometer analysis. Data analysis on the specimen are in progress. © 2013 European Space Agency.

Hair root cells actively divide in a hair follicle, and they sensitively reflect physical conditions. By analyzing the human hair, we can know stress levels on the human body and metabolic conditions caused by microgravity environment and cosmic radiation. The Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples of astronauts who stayed in the International Space Station (ISS) for 6 months. During long-term flights, the physiological effects on astronauts include muscle atrophy and bone calcium loss. Furthermore, radiation and psychological effects are important issue to consider. Therefore, an understanding of the effects of the space environment is important for developing countermeasures against the effects experienced by astronauts. In this experiment, we identify functionally important target proteins that integrate transcriptome, mineral metabolism and proteome profiles from human hair. To compare the protein expression data with the gene expression data from hair roots, we developed the protein processing method. We extracted the protein from five strands of hair using ISOGEN reagents. Then, these extracted proteins were analyzed by LC-MS/MS. These collected profiles will give us useful physiological information to examine the effect of space flight. © 2013 European Space Agency.

In the space environment, physiological alterations, such as low bone density, muscle weakness and decreased immunity, are caused by microgravity and cosmic radiation. On the other hand, it is known that the leg muscles are hypertrophy by 2G-gravity. An understanding of the effects on human body from microgravity to hyper-gravity is very important. Recently, the Japan Aerospace Exploration Agency (JAXA) has started a project to detect the changes on gene expression and mineral metabolism caused by microgravity by analyzing the hair of astronauts who stay in the international Space Station (ISS) for a long time. From these results of human hair's research, the genetic effects of human hair roots by microgravity will become clear. However, it is unclear how the gene expression of hair roots was effected by hypergravity. Therefore, in this experiment, we analyzed the effect on mice skin contained hair roots by comparing microgravity or hypergravity exposed mice. The purpose of this experiment is to evaluate the genetic effects on mice skin by microgravity or 2G-gravity. The samples were taken from mice exposed to space flight (FL) or hypergravity environment (2G) for 3-months, respectively. The extracted and amplified RNA from these mice skin was used to DNA microarray analysis. in this experiment, we analyzed the effect of gravity by using mice skin contained hair roots, which exposed space (FL) and hyper-gravity (2G) for 3 months and each control. By DNA microarray analysis, we found the common 98 genes changed in both FL and 2G. Among these 98 genes, the functions and pathways were identified by Gene Ontology (GO) analysis and Ingenuity Pathways Analysis (IPA) software. Next, we focused the one of the identified pathways and compared the effects on each molecules in this pathways by the different environments, such as FL and 2G. As the results, we could detect some interesting molecules, which might be depended on the gravity levels. In addition, to investigate the relationships between genes and protein expression, the proteome analysis was performed. From the result of 2-dimentional electrophoresis, we could detect the some different spots between FL and 2G. These identifications are now in progress using by MALDI-TOF-MS/MS. These results suggested that many genes or proteins on the mice skin might be effected by the different gravity levels. © 2013 European Space Agency.

How microgravitational space environments affect aging is not well understood. We observed that, in Caenorhabditis elegans, spaceflight suppressed the formation of transgenically expressed polyglutamine aggregates, which normally accumulate with increasing age. Moreover, the inactivation of each of seven genes that were down-regulated in space extended lifespan on the ground. These genes encode proteins that are likely related to neuronal or endocrine signaling: acetylcholine receptor, acetylcholine transporter, choline acetyltransferase, rhodopsin-like receptor, glutamate-gated chloride channel, shaker family of potassium channel, and insulin-like peptide. Most of them mediated lifespan control through the key longevity-regulating transcription factors DAF-16 or SKN-1 or through dietary-restriction signaling, singly or in combination. These results suggest that aging in C. elegans is slowed through neuronal and endocrine response to space environmental cues.

Kohno S, Yamashita Y, Abe T, Hirasaka K, Oarada M, Ohno A, Teshima-Kondo S, Higashibata A, Choi I, Mills EM, Okumura Y, Terao J, Nikawa T. Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages. J Appl Physiol 112: 1773-1782, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00103.2012.- Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-alpha and interleukin-1 beta, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.

Background: Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. Methods: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80 degrees C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. Results: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific alpha-actin protein in both spaceflight and GC conditions. Conclusions: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.

Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages. Muscle Nerve 43: 223-229, 2011

In response to a change in the direction of gravity, morphogenetic changes of fruiting bodies of fungi are usually observed as gravitropism. Although gravitropism in higher fungi has been studied for over 100 years, there is no convincing evidence regarding the graviperception mechanism in mushrooms. To understand gravitropism in mushrooms, we isolated differentially expressed genes in Pleurotus ostreatus (oyster mushroom) fruiting bodies developed under three-dimensional clinostat-simulated microgravity. Subtractive hybridization, cDNA representational difference analysis was used for gene analysis and resulted in the isolation of 36 individual genes (17 upregulated and 19 downregulated) under clinorotation. The phenotype of fruiting bodies developed under simulated microgravity vividly depicted the gravitropism in mushrooms. Our results suggest that the differentially expressed genes responding to gravitational change are involved in several potential cellular mechanisms during fruiting body formation of P. ostreatus.

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.

Oil Earth, it is common to employ laboratory animals such as the nematode Caenorhabditis elegans to help understand human health concerns. Similar studies in Earth orbit should help understand and address the concerns associated with spaceflight. The "International Caenorhabditis elegans Experiment FIRST" (ICE FIRST), was carried out onboard the Dutch Taxiflight in April of 2004 by all international collaboration of laboratories in France, Canada, Japan and the United States. With the exception of a slight movement defect upon return to Earth, the result of altered muscle development, no significant abnormalities were detected in spaceflown C. elegans. Work from Japan revealed apoptosis proceeds normally and work from Canada revealed no significant increase in the rate of mutation. These results suggest that C elegans call be used to study non-lethal responses to spaceflight and call possibly be developed as a biological sensor. To further our understanding of C elegans response to spaceflight, we examined the gene transcription response to the 10 days in space using a near full genome microarray analysis. The transcriptional response is consistent with the observed normal developmental timing, apoptosis, DNA repair, and altered muscle development. The genes identified as altered in response to spaceflight are enriched for genes known to be regulated, in C elegans, in response to altered environmental conditions (Insulin and TGF-beta regulated). These results demonstrate C elegans call be used to study the effects of altered gravity and suggest that C. elegans responds to spaceflight by altering the expression of at least some of the same metabolic genes that are altered in response to differing terrestrial environments. Published by Elsevier Ltd. oil behalf of COSPAR.

first International Caenorhabditis elegans Experiment (ICE-First) was carried out using a Russian Soyuz spacecraft from April 19-30, 2004. This experiment was a part of the program of the DELTA (Dutch Expedition for Life science Technology and Atmospheric research) mission, and the space agencies that participate in the International Space Station (ISS) program formed international research teams. A Japanese research team that conducted by Japan aerospace Exploration Agency (JAXA) investigated the following aspects of the organism: (1) whether meiolic chromosomal dynamics and apoptosis in the germ cells were normal under microgravity conditions, (2) the effect of the space flight on muscle cell development, and (3) the effect of the space flight on protein aggregation. In this article, we summarize the results of these biochemical and molecular biological analyses.

The molecular mechanisms underlying muscle atrophy during spaceflight are not well understood. We have analyzed the effects of a 10-day spaceflight on Caenorhabditis elegans muscle development. DNA microarray, real-time quantitative PCR, and quantitative western blot analyses revealed that the amount of MHC in both body-wall and pharyngeal muscle decrease in response to spaceflight. Decreased transcription of the body-wall myogenic transcription factor HLH-1 ( CeMyoD) and of the three pharyngeal myogenic transcription factors, PEB-1, CEH-22 and PHA-4 were also observed. Upon return to Earth animals displayed reduced rates of movement, indicating a functional defect. These results demonstrate that C. elegans muscle development is altered in response to spaceflight. This altered development occurs at the level of gene transcription and was observed in the presence of innervation, not simply in isolated cells. This important finding coupled with past observations of decreased levels of the same myogenic transcription factions in vertebrates after spaceflight raises the possibility that altered muscle development is a contributing factor to spaceflight-induced muscle atrophy in vertebrates.

Background: The irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells. Results: Clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small GTPase Rho. Actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. From the gene and protein expression analyses, we found that the expression level of leukemia-associated Rho guanine nucleotide exchange factor (LARG), which activates Rho, was downregulated under clinorotation. Moreover, we identified the full-length LARG cDNA. The amount of GTP-bound RhoA, that is, the active form of RhoA, decreased under this condition. Conclusion: The activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D) clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF) family, was identified, and its gene expression was observed to be downregulated under clinorotation. This downregulation subsequently resulted in the repression of RhoA activation. These results indicated that the disorganization of the actin fibers was caused by the inhibition of Rho activation by 3D clinorotation. © 2006 Higashibata et al licensee BioMed Central Ltd.

The effects of hindlimb unloading and recovery with or without running exercise on morphological and metabolic properties of soleus muscle fibers and their spinal motoneurons in rats were investigated. Ten-week-old rats were hindlimb suspended for 2 weeks and thereafter were rehabilitated with or without voluntary running exercise for 2 weeks. A decreased percentage of type I fibers and atrophy of all types of fibers were observed after hindlimb unloading. In addition, decreased oxidative enzyme activity of all types of fibers was observed after hindlimb unloading. In contrast, an improvement in the decreased percentage of type I fibers, decreased fiber cross-sectional area, and decreased fiber oxidative enzyme activity was observed after recovery with running exercise, but not without running exercise. There were no changes in the number, cell body size, or oxidative enzyme activity of motoneurons innervating the soleus muscle after hindlimb unloading or recovery with or without running exercise. These results indicate that running exercise is beneficial for the recovery of the decreased percentage of type I fibers and the atrophy and decreased oxidative enzyme activity of all types of fibers in the soleus muscle induced by hindlimb unloading and that there are no changes in morphological or metabolic properties of spinal motoneurons innervating the soleus muscle following decreased or increased neuromuscular activity. (C) 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved.

Cell body sizes and oxidative enzyme (succinate dehydrogenase) activities of spinal motoneurons innervating the soleus muscle were determined in rats ranging in postnatal age from 3 to 13 weeks. The soleus motoneurons were labeled by a retrograde neuronal tracer, nuclear yellow. The mean cell body sizes of motoneurons increased from 3 to 7 weeks of age, while the mean succinate dehydrogenase activities of motoneurons decreased from 3 to 7 weeks of age. There were no changes in mean cell body size or mean succinate dehydrogenase activity of motoneurons from 7 to 13 weeks of age. An inverse relationship between cell body size and succinate dehydrogenase activity of motoneurons was observed, irrespective of age. These results indicate that motoneurons innervating the rat soleus muscle show the adult pattern of cell body size and succinate dehydrogenase activity at an earlier stage of postnatal growth, 7 weeks of age. (C) 2003 ISDN. Published by Elsevier Ltd. All rights reserved.