NASDA aquatic animal experiment facilities for Space Shuttle.

National Space Development Agency of Japan (NASDA) has been developed aquatic animal experiment facilities for space experiments using NASA Space Shuttle. Vestibular Function Experiment Unit (VFEU) has been firstly designed and developed for Spacelab-J mission (STS-47), and 8 days space experiment with carp has been performed. Following, the VFEU, Aquatic Animal Experiment Unit (AAEU) has been developed to accommodate small aquatic animals second International Microgravity Laboratory mission (IML-2, STS-65). Four kinds of space experiments with goldfish, medaka, newt, and newt eggs have been performed for 15 days mission duration. Then, VFEU has been improved to accommodate marine fish under low temperature condition for Neurolab (STS-90) and STS-95 missions. 17 days (STS-90) and 9 days (STS-95) experiments with oyster toadfish have been performed by using the VFEU. This report summarizes the outline of these aquatic animal experiment facilities.     

VFEU water quality control in STS-95 mission.

In STS-95 Space Shuttle mission, an aquatic animal research facility, Vestibular Function Experiment Unit (VFEU), was flown to perform neurobiological experiment with marine fish, oyster toadfish. For this purpose, we have developed a sea water purification system using highly active nitrifying bacteria at low temperature. With this system, the water quality in the VFEU was maintained in sufficient condition to keep the toadfish in healthy state for 9 days of the mission. This report summarizes the efficiency of the filter system based on the results from pre-flight bacterial preparation, water analysis of samples taken during flight, and the post-flight analysis of the bacterial filter.     

Water quality management for low temperature marine fishes in space.

Vestibular Function Experiment Unit (VFEU), one of the Spacelab facility flown in Neurolab mission (STS-90) in April, 1998, was to support neurophysiological research using a marine fish, Opsanus tau (oyster toadfish). The functions of the VFEU were primarily a quality management of environmental water during the mission at 14 degrees C and for acquiring physiological signals from implanted micro-electrodes in the otolith nerves as well as the spatial acceleration of the fish. A key element of the life support system was a balanced biological filter containing two types of nitrifying bacteria, Nitrosomonas for ammonia oxidization and Nitrobacter for nitrite oxidization. Although the 16 days mission was successful, two toadfishes died in late phase of the mission. Ammonium concentration in those two life support systems elevated to remarkably high level at the end of the mission whereas the other two indicated very low. This report summarizes the results of the water quality management of the VFEU during the Neurolab mission based on analysis of water samples taken during the flight and those taken prior and just after the flight.     

What kind of eye movements will goldfish show in space?

Six goldfish (1 normal, 1 with otoliths removed on both sides, 4 with otoliths removed on one side) were flown in space. The behaviors of the fish were recorded with a video camera on Mission Elapsed Time (MET) Day-00, 02, 05, 08, and 12. On MET Day-00, fish with otoliths removed on one side showed rolling behavior toward the operated side. No rolling behaviors were observed after MET Day-8. Five fish showed backward looping behaviors during the mission. After the space experiment, torsional eye movements and vertical eye movements were examined by body tilting. The experiments showed that the sensitivity of eye movements were low for head up tilting and tilting to operated side.     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

Hypergravity studies in the Netherlands

It looks like that with the utilization phase of the International Space Station (ISS) scientists will have the possibility to perform long duration and more sophisticated microgravity experiments than could be performed previously. In preparation for these spaceflight studies, ground based experiment tools for simulated (or real) microgravity and hypergravity are important. To provide the infrastructure and user support necessary to perform these ground based studies we have setup the Dutch Experiment Support Center, DESC. This paper will focus on the three Dutch centrifuge facilities. It is shown that these hypergravity facilities can be used to show sounding rocket launch effects, identify alterations in body mass, bone parameters and matrix composition in rodents as well as to derive a test protocol for the Space Adaptation Syndrome in humans. DESC coordinates the use of these centrifuge facilities.     

The BIORACK facility and its performance during the IML-2 Spacelab mission

The configuration and performance of the Biorack facility during the Second International Microgravity Laboratory mission (IML-2; 8-23 July 1995) is described in detail. During this Spacelab mission, Biorack flew with two incubators (22 degrees C and 37 degrees C), glovebox, cooler (5 degrees C) and four passive thermal conditioning units (PTCU; 5 degrees C and 10 degrees C) in the stowage. The crew worked more than 40 h to perform 19 Biorack experiments originating from seven European countries. Almost 200 Biorack experiment containers had to be translocated in about 1500 predetermined steps before the Space Shuttle Columbia returned after nearly 14 days: 18 h or 236 orbits in space to Kennedy Space Center, Florida.     

Mechanism of vestibular adaptation of fish under microgravity.

In a space experiment, the adaptation of goldfish behavior during flight and readaptation after landing were investigated. Six goldfish (1 normal, 1 with otoliths removed on both sides, 4 with otoliths removed on one side) were flown in a fish package (F/P) of Aquatic Animal Experiment Unit (AAEU). The dorsal light responses (DLRs) of fish with otoliths removed were recorded after operation until launch and after landing. The behaviors of the fish were recorded with a video camera on Mission Elapsed Time (MET) Day-00, 02, 05, 08, 12. On MET Day-00, two fish with otoliths removed on one side showed flexion of body toward the operated side. These fish also showed rolling behavior toward the operated side. However, the body flexion disappeared on MET Day-05 or MET Day-08. No rolling behaviors were observed after that time. Five fish showed backward looping behaviors during the mission. Although the frequency of looping episodes decreased after MET Day-08, five fish still showed looping behavior on MET Day-12, that was the last day of video recording on orbit. In microgravity, visual system of fish did not seem to provide sufficient cues to prevent them from looping or rolling. After landing, no looping and rolling behavior was observed. However, the tilt angle of the DLR increased in the fish with otolith removed 5 month before launch but not in normal fish and those with otoliths removed 2 weeks before launch. These results suggest that the behavioral dysfunction and the adaptational process in space are dependent on vestibular inputs.     

Preventing annoyance from odors in spaceflight: a method for evaluating the sensory impact of rodent housing.

For the scientific community, the ability to fly mice under weightless conditions in space offers several advantages over the use of rats. These advantages include the option of testing a range of transgenic animals, the ability to increase the number of animals that can be flown, and reduced demands on shuttle resources (food, water, animal mass) and crew time (for water refill). Mice have been flown in animal enclosure module (AEM) hardware only once [Space Shuttle Transport System (STS)-90] and were dissected early in the mission, whereas rats have been flown in the AEM on >20 missions. This has been due, in part, to concerns that strong and annoying odors from mouse urine (vs. rat urine) will interfere with crew performance in the shuttle middeck. To screen and approve mice for flight, a method was developed to evaluate the odor containment performance of AEMs housing female C57BL/6J mice compared with AEMs housing Sprague-Dawley rats across a 21-day test period. Based on the results of this test, consensus was reached that mice could fly in the AEM hardware for up to 17 days (including prelaunch and contingency) and that the AEM hardware would likely contain odors beyond this duration. Human sensory and electronic nose analysis of the AEMs postflight demonstrated their success in containing odors from mice for the mission duration of STS-108 (13 days). Although this paper focuses specifically on odor evaluations for the space shuttle, the concern is applicable to any confined, closed-system environment for human habitation.     

Preparatory studies for the use of plant protoplasts in space research

An experiment using plant protoplasts has been accepted for the IML-1 mission to be flown on a space shuttle in 1991. Preparatory experiments include studies of cell wall formation, cell division, the effect of simulated weightlessness using fast and slow rotating clinostats, and the development and testing of hardware for the IML-1 mission. After 24 h at 25°C, protoplasts isolated from hypocotyls or leaves of rapeseed seedlings, or from carrot suspension cells, show 60, 20 and 15% cell wall formation, respectively. The time course of formation of the cell wall and cell division could be delayed by treatment at low temperatures or immobilization in alginate or agarose. This aspect is of importance in connection with problems of late access to the space shuttle before launch. At 4°C only 18% of the rapeseed hypocotyl protoplasts had formed cell walls after 24 h. Protoplasts immobilised in agarose or alginate gradually regain their cell division capacity and after 72 h the frequencies are 51 and 26%, respectively, compared to non-immobilised control protoplasts. A significant decrease in cell division activity is observed after rotation for 6 h on the slow clinostat. A similar effect is not observed on the fast clinostat. Protoplasts, cultured in the specially designed plant chamber for up to 14 days established cell aggregates which have further developed into plants.     

Nastic curvatures of wheat coleoptiles that develop in true microgravity

Dark-grown wheat coleoptiles developed strong curvatures within 5 h of being transferred in orbit from a 1 g centrifuge to microgravity during an experiment flown on the IML-1 shuttle mission. The curving tendency was strongest in seedlings that were immature, with coleoptiles shorter than 10 mm at the time of transfer. The curvature direction was non-random, and directed away from the caryopsis (the coleptile face adjacent to the caryopsis becoming convex). The curvatures were most marked in the basal third of the coleoptiles, contrasting with phototropic responses, which occur in the apical third. We interpret these curvatures as being nastic, and related to the curvatures commonly reported to occur during clinostat rotation treatments.     

Activation of nervous system development genes in bone marrow derived mesenchymal stem cells following spaceflight exposure

Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity‐induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA‐8 + Increment 13) from March 30 to April 8, 2006 (experiment “Stroma‐2”). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo‐inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell‐cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC. J. Cell. Biochem. 111: 442–452, 2010. © 2010 Wiley‐Liss, Inc.     

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.     

Effect of spaceflight on oxidative and antioxidant enzyme activity in rat diaphragm and intercostal muscles

There are limited data regarding changes in oxidative and antioxidant enzymes induced by simulated or actual weightlessness, and any additional information would provide insight into potential mechanisms involving other changes observed in muscles from animals previously flown in space. Thus, the NASA Biospecimen Sharing Program was an opportunity to collect valuable information. Oxidative and antioxidant enzyme levels, as well as lipid perioxidation, were measured in respiratory muscles from rats flown on board Space Shuttle mission STS-54. The results indicated that there was an increasing trend in citrate synthase activity in the flight diaphragm when compared to ground based controls, and there were no significant changes observed in the intercostal muscles for any of the parameters. However, lipid peroxidation was significantly (p<0.05) decreased in the flight diaphragm. These results indicate that 6 day exposure to microgravity may have a different effect on oxidative and antioxidant activity in rat respiratory muscles when compared to data from previous 14 day hindlimb suspension studies.     

BIOLAB, EPU and EMCS for cell culture experiments on the ISS

Two ESA facilities are under development for biological research on the International Space Station: BIOLAB as part of the European "Columbus" Laboratory and the European Modular Cultivation System (EMCS), foreseen for accommodation in the US Lab "Destiny". Both facilities have an incubator (18-40 degrees C) and use standard Experiment Containers, mounted on two centrifuge rotors providing either microgravity or variable g-levels from 0.001 x g to 2.0 x g. Standard interface plates supply each container with power and data lines, with gas (controlled CO2, O2 and water vapour concentration; trace gas removal), and--for EMCS only--with water. The degree of automation is higher in BIOLAB: it contains a robotic Handling Mechanism for automatic sampling and handling of liquids, which can be stored at cool or cold temperatures or injected for automatic on-board analysis into a microscope or a spectrophotometer. For analyses on the running centrifuge, small automatic microscopes can be installed in the Experiment Containers. Several designs for supporting cell culture experiments have been studied for BIOLAB and EMCS. BIOLAB has in addition a Bio-Glovebox, which can be sterilised and where new cell cultures may be prepared under 1 x g conditions from deep-frozen samples in the Experiment Preparation Unit (EPU): the cryo-protectant will be removed by automatic washing cycles. Both facilities, EMCS and BIOLAB (with EPU), have also provisions for telescience operations through video, data and command lines, either operated by the crew or by the experimenter on ground.     

Transcriptional analysis of normal human fibroblast responses to microgravity stress

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.     

Histomorphometric study of tibia of rats exposed aboard American Spacelab Life Sciences 2 Shuttle Mission

Tibial bones of rats flown onboard the SLS-2 shuttle mission were studied. Trabecular bone parameters were investigated, including growth plate height, trabecular bone volume, thickness and number, and trabecular separation in the primary and secondary spongiosa. Several histomorphometric changes were noted, allowing researchers to conclude that exposure to microgravity resulted in osteopenia of spongy bone of tibial metaphysis. The roles of bone formation and bone resorption are discussed.     

Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.     

Inactivation of Poliovirus Type 1 by the Kelly-Purdy Ultraviolet Seawater Treatment Unit

Three experiments were conducted to determine the effect of ultraviolet (UV) radiation on poliovirus-contaminated seawater. In two of the experiments, the effectiveness of the Kelly-Purdy UV Seawater Treatment Unit to inactivate poliovirus type 1 (T(1)) suspended in continuously flowing seawater was determined. In experiment 1, the observed survival ratio of poliovirus T(1) was 2.3 x 10(-4) (99.98% reduction) in 15.7 sec. No virus was detected (<0.2 plaque-forming unit/ml) in 20.6 seconds. The calculated half-life value was 1.29 sec. In experiment 2, the observed survival ratio of poliovirus T(1) was 5.9 x 10(-4) (99.94% reduction) in 11.7 sec. No virus was detected in 15.7 sec. The calculated half-life value was 1.37 sec. In experiment 3, a laboratory-controlled UV experiment designed to closely simulate the geometry of the continuously flowing seawater system, the observed survival ratios of poliovirus T(1) were 9.7 x 10(-3) (99.03% reduction) and 3.6 x 10(-4) (99.96% reduction) in 15 and 30 sec, respectively; the calculated half-life value was 2.38 sec. A statistically significant difference was found between the inactivation rates of poliovirus T(1) in the two test systems. This rate difference was attributed primarily to UV dosage and stirring effects. The data indicated that UV radiation effectively inactivated poliovirus T(1) in flowing seawater. These results validate the efficacy of the Kelly-Purdy UV Seawater Treatment Unit for use in commercial depuration systems.     

STS-95 Space Experiments (plants and cell biology).

We report the outline of Space Experiments conducted on Space Shuttle (STS-95) launched in autumn of 1998. In this STS-95 mission, Japanese astronaut Dr. Chiaki Mukai achieved her 2nd space flight and conducted a part of 82 space experiments including Japanese experiments. US astronaut Senator John Glenn also achieved his second space flight, 36 years after his first space flight. Senator Glenn was a leader of the original (the first) 7 US astronauts and very famous in US because he succeeded US first orbital space flight around the earth. NASDA had started the project of space experiment using STS-95 at the summer of 1997, therefore we had only one year for the all preparation Yamashita, et al. Biological Sciences in Space, Vol.12 No.3(1998). Scientific results will be reported by investigators, therefore we report here how we had been developing the space experiment plan, on board operation procedure and ground operations including ground control experiments about four plant experiments and one cell biology experiment.