Preliminary characterization of persisting circadian rhythms during space flight.

In order to evaluate the function of the circadian timing system in space, the circadian rhythm of conidiation of the fungus Neurospora crassa was monitored in constant darkness on the STS 9 flight of the Space Shuttle Columbia. During the first 7 days of spaceflight many tubes showed a marked reduction in the apparent amplitude of the conidiation rhythm, and some cultures appeared arrhythmic. There was more variability in the growth rate and circadian rhythms of individual cultures in space than is usually seen on earth. The results of this experiment indicate that while the circadian rhythm of Neurospora conidiation can persist outside of the Earth's environment, either the timekeeping process or its expression is altered in space.     

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).     

Gravitational force regulates elongation growth of Arabidopsis hypocotyls by modifying xyloglucan metabolism.

Growth of dark-grown Arabidopsis hypocotyls was suppressed under hypergravity conditions (300 g), or was stimulated under microgravity conditions in space (Space Shuttle STS-95). The mechanical extensibility of cell walls decreased and increased under hypergravity and microgravity conditions, respectively. The amounts of cell wall polysaccharides (pectin, hemicellulose-I, hemicellulose-II and cellulose) per unit length of hypocotyls increased under hypergravity conditions, and decreased under microgravity conditions. The amount and the molecular mass of xyloglucans also increased under the hypergravity conditions, while those decreased under microgravity conditions. The activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls decreased and increased under hypergravity and microgravity conditions, respectively. These results indicate that the amount and the molecular mass of xyloglucans are affected by the magnitude of gravity and that such changes are caused by changes in xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell walls by gravity stimulus may be the primary event determining the cell wall extensibility, thereby regulating the growth rate of Arabidopsis hypocotyls.     

Effects of space flight and IGF-1 on immune function

We tested the hypothesis that insulin-like growth factor-1 (IGF-1) would ameliorate space flight-induced effects on the immune system. Twelve male, Sprague-Dawley rats, surgically implanted with mini osmotic pumps, were subjected to space flight for 10 days on STS-77. Six rats received 10 mg/kg/day of IGF-1 and 6 rats received saline. Flight animals had a lymphocytopenia and granulocytosis which were reversed by IGF-1. Flight animals had significantly higher corticosterone levels than ground controls but IGF-1 did not impact this stress hormone. Therefore, the reversed granulocytosis did not correlate with serum corticosterone. Space flight and IGF-1 also combined to induce a monocytopenia that was not evident in ground control animals treated with IGF-1 or in animals subjected to space flight but given physiological saline. There was a significant increase in spleen weights in vivarium animals treated with IGF-1, however, this change did not occur in flight animals. We observed reduced agonist-induced lymph node cell proliferation by cells from flight animals compared to ground controls. The reduced proliferation was not augmented by IGF-1 treatment. There was enhanced secretion of TNF, IL-6 and NO by flight-animal peritoneal macrophages compared to vivarium controls, however, O₂ secretion was not affected. These data suggest that IGF-1 can ameliorate some of the effects of space flight but that space flight can also impact the normal response to IGF-1.     

Approaches in the determination of plant nutrient uptake and distribution in space flight conditions.

The effective growth and development of vascular plants rely on the adequate availability of water and nutrients. Inefficiency in either the initial absorption, transportation, or distribution of these elements are factors which impinge on plant structure and metabolic integrity. The potential effect of space flight and microgravity conditions on the efficiency of these processes is unclear. Limitations in the available quantity of space-grown plant material and the sensitivity of routine analytical techniques have made an evaluation of these processes impractical. However, the recent introduction of new plant cultivating methodologies supporting the application of radionuclide elements and subsequent autoradiography techniques provides a highly sensitive investigative approach amenable to space flight studies. Experiments involving the use of gel based 'nutrient packs' and the radionuclides calcium-45 and iron-59 were conducted on the Shuttle mission STS-94. Uptake rates of the radionuclides between ground and flight plant material appeared comparable.     

Production and action of cytokines in space.

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.     

Adaptations of the vestibular system to short and long-term exposures to altered gravity.

Long-term space flight creates unique environmental conditions to which the vestibular system must adapt for optimal survival of a given organism. The development and maintenance of vestibular connections are controlled by environmental gravitational stimulation as well as genetically controlled molecular interactions. This paper describes the effects of hypergravity on axonal growth and dendritic morphology, respectively. Two aspects of this vestibular adaptation are examined: (1) How does long-term exposure to hypergravity affect the development of vestibular axons? (2) How does short-term exposure to extremely rapid changes in gravity, such as those that occur during shuttle launch and landing, affect dendrites of the vestibulocerebellar system? To study the effects of longterm exposures to altered gravity, embryonic rats that developed in hypergravity were compared to microgravity-exposed and control rats. Examination of the vestibular projections from epithelia devoted to linear and angular acceleration revealed that the terminal fields segregate differently in rat embryos that gestated in each of the gravitational environments.To study the effects of short-term exposures to altered gravity, mice were exposed briefly to strong vestibular stimuli and the vestibulocerebellum was examined for any resulting morphological changes. My data show that these stimuli cause intense vestibular excitation of cerebellar Purkinje cells, which induce up-regulation of clathrin-mediated endocytosis and other morphological changes that are comparable to those seen in long-term depression. This system provides a basis for studying how the vestibular environment can modify cerebellar function, allowing animals to adapt to new environments.     

Seedling growth and development on space shuttle.

Young pine seedlings, and mung bean and oat seeds were flown on shuttle flights, STS-3 and STS-51F, in March, 1982 and July/August, 1985, respectively. The plant growth units built to support the two experiments functioned mechanically as anticipated and provided the necessary support data. Pine seedlings exposed to the microgravity environment of the space shuttle for 8 days continued to grow at a rate similar to ground controls. Pine stems in flight seedlings, however, averaged 10 to 12% less lignin than controls. Flight mung beans grew slower than control beans and their stems contained about 25% less lignin than control seedlings. Reduced mung bean growth in microgravity was partly due to slower germination rate. Lignin also was reduced in flight oats as compared to controls. Oats and mung beans exhibited upward growing roots which were not observed in control seedlings. Chlorophyll A/B ratios were lower in flight tissues than controls. The sealed PGCs exhibited large variations in atmospheric gas composition but the changes were similar between flight and ground controls. Ethylene was present in low concentrations in all chambers.     

Influence of gravity on the circadian timing system.

The circadian timing system (CTS) is responsible for daily temporal coordination of physiological and behavioral functions both internally and with the external environment. Experiments in altered gravitational environments have revealed changes in circadian rhythms of species ranging from fungi to primates. The altered gravitational environments examined included both the microgravity environment of spaceflight and hyperdynamic environments produced by centrifugation. Acute exposure to altered gravitational environments changed homeostatic parameters such as body temperature. These changes were time of day dependent. Exposure to gravitational alterations of relatively short duration produced changes in both the homeostatic level and the amplitude of circadian rhythms. Chronic exposure to a non-earth level of gravity resulted in changes in the period of the expressed rhythms as well as in the phase relationships between the rhythms and between the rhythms and the external environment. In addition, alterations in gravity appeared to act as a time cue for the CTS. Altered gravity also affected the sensitivity of the pacemaker to other aspects of the environment (i.e., light) and to shifts of time cues. Taken together, these studies lead to the conclusion that the CTS is indeed sensitive to gravity and its alterations. This finding has implications for both basic biology and space medicine.     

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity.

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.     

Radiation measurements on the flight of IML-2.

The second flight of the International Microgravity Laboratory (IML-2) on Space Shuttle flight STS-65 provided a unique opportunity for the intercomparison of a wide variety of radiation measurement techniques. Although this was not a coordinated or planned campaign, by sheer chance, a number of space radiation experiments from several countries were flown on this mission. There were active radiation measuring instruments from Japan and US, and passive detectors from US, Russia, Japan, and Germany. These detectors were distributed throughout the Space Shuttle volume: payload bay, middeck, flight deck, and Spacelab. STS-65 was launched on July 8, 1994, in a 28.45 degrees x 306 km orbit for a duration of 14 d 17 hr and 55 min. The crew doses varied from 0.935 mGy to 1.235 mGy. A factor of two variation was observed between various passive detectors mounted inside the habitable Shuttle volume. There is reasonable agreement between the galactic cosmic ray dose, dose equivalent and LET spectra measured by the tissue equivalent proportional counter flown in the payload bay with model calculations. There are significant differences in the measurements of LET spectra measured by different groups. The neutron spectrum in the 1-20 MeV region was measured. Using fluence-dose conversion factors, the neutron dose and dose equivalent rates were 11 +/- 2.7 microGy/day and 95 +/- 23.5 microSv/day respectively. The average east-west asymmetry of trapped proton (>3OMeV) and (>60 MeV) dose rate was 3.3 and 1.9 respectively.     

Effects of simulated weightlessness on fish otolith growth: Clinostat versus Rotating-Wall Vessel

Stimulus dependence is a general feature of developing sensory systems. It has been shown earlier that the growth of inner ear heavy stones (otoliths) of late-stage Cichlid fish (Oreochromis mossambicus) and Zebrafish (Danio rerio) is slowed down by hypergravity, whereas microgravity during space flight yields an opposite effect, i.e. larger than 1 g otoliths, in Swordtail (Xiphophorus helleri) and in Cichlid fish late-stage embryos. These and related studies proposed that otolith growth is actively adjusted via a feedback mechanism to produce a test mass of the appropriate physical capacity. Using ground-based techniques to apply simulated weightlessness, long-term clinorotation (CR; exposure on a fast-rotating Clinostat with one axis of rotation) led to larger than 1 g otoliths in late-stage Cichlid fish. Larger than normal otoliths were also found in early-staged Zebrafish embryos after short-term Wall Vessel Rotation (WVR; also regarded as a method to simulate weightlessness). These results are basically in line with the results obtained on Swordtails from space flight.     

Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles (Xenopus laevis).

In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1)the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) microgravity induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.     

Circadian rhythms in a long-term duration space flight.

In order to maintain cosmonaut health and performance, it is important for the work-rest schedule to follow human circadian rhythms (CR). What happens with CR in space flight? Investigations of CR in mammals revealed, that the circadian phase in flight is less stable, probably due to a displacement of the range of entrainment, resulting from internal period change (the latter was confirmed on insects). The circadian period may be a gravity-dependent parameter. If so, the basic biological requirement for the day length might be different in weightlessness. On this basis, a higher risk of desynchronosis is expected in a long-duration space flight. As a countermeasure, a non-24-hr day length could be suggested, being close to the internal circadian period (in humans about 25 hr). Taking into account a possible displacement of period in weightlessness, it seems reasonable to establish a flexible work-rest schedule, capable to follow the body temperature CR by means of biofeedback.     

First flight of the ASTROCULTURE (TM) experiment as a part of the U.S. Shuttle/MIR program.

A number of space-based experiments have been conducted to assess the impact of microgravity on plant growth and development. In general, these experiments did not identify any profound impact of microgravity on plant growth and development, though investigations to study seed development have indicated difficulty in plants completing their reproductive cycle. However, it was not clear whether the lack of seed production was due to gravity effects or some other environmental condition prevailing in the unit used for conducting the experiment. The ASTROCULTURE (TM) flight unit contains a totally enclosed plant chamber in which all the critically important environmental conditions are controlled. Normal wheat (Triticum aestivum L.) growth and development in the ASTROCULTURE (TM) flight unit was observed during a ground experiment conducted prior to the space experiment. Subsequent to the ground experiment, the flight unit was transported to MIR by STS-89, as part of the U.S. Shuttle/MIR program, in an attempt to determine if super dwarf wheat plants that were germinated in microgravity would grow normally and produce seeds. The experiment was initiated on-orbit after the flight unit was transferred from the Space Shuttle to MIR. The ASTROCULTURE (TM) flight unit performed nominally for the first 24 hours after the flight unit was activated, and then the unit stopped functioning abruptly. Since it was not possible to return the unit to nominal operation it was decided to terminate the experiment. On return of the flight unit, it was confirmed that the control computer of the ASTROCULTURE (TM) flight unit sustained a radiation hit that affected the control software embedded in the computer. This experience points out that at high orbital inclinations, such as that of MIR and that projected for the International Space Station, the danger of encountering harmful radiation effects are likely unless the electronic components of the flight hardware are resistant to such impacts.     

The ASTROCULTURE(TM) flight experiment series, validating technologies for growing plants in space.

A flight experiment, ASTROCULTURE(TM)-1 (ASC-1), to evaluate the operational characteristics and hardware performance of a porous tube nutrient delivery system (PTNDS) was flown on STS-50 as part of the U.S. Microgravity Laboratory-1 mission, 25 June to 9 July, 1992. This experiment is the first in a series of planned ASTROCULTURE(TM) flights to validate the performance of subsystems required to grow plants in microgravity environments. Results indicated that the PTNDS was capable of supplying water and nutrients to plants in microgravity and that its performance was similar in microgravity to that in 1g on Earth. The data demonstrated that water transfer rates through a rooting matrix are a function of pore size of the tubes, the degree of negative pressure on the 'supply' fluid, and the pressure differential between the 'supply' and 'recovery' fluid loops. A slightly greater transfer rate was seen in microgravity than in 1g, but differences were likely related to the presence of hydrostatic pressure effects at 1g. Thus, this system can be used to support plant growth in microgravity or in partial gravity as on a lunar or Mars base. Additional subsystems to be evaluated in the ASTROCULTURE(TM) flight series of experiments include lighting, humidity control and condensate recovery, temperature control, nutrient composition control, CO2 and O2 control, and gaseous contaminant control.     

The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.     

The "gaseous" environment in sealed BRIC-100VC canisters flown on 'Mir' with embryogenic daylily cell cultures.

As part of the "Cellular Mechanisms of Spaceflight-Specific Stress to Plants" experiment, nine BRIC (Biological Research in Canisters) 100VC canisters, each containing four 100 mm dia polycarbonate petri dishes with embryogenic daylily (Hemerocallis sp.) cultures, were launched on 12 Jan 97 (STS-81), transferred to 'Mir' and returned on 24 May 97 (STS-84). Pre-flight, flight and ground control data for temperature, relative humidity, CO2 and ethylene in the BRIC canisters are presented.     

Effects of space flight on surface marker expression.

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.     

Biorhythms on cellular and organismic level (introduction).

The regular change of day and night, of light and darkness during millions of years has strongly affected the development of life on earth. Many organisms adapted themselves to this environmental condition and, finally, evolved an endogenous timer which usually is in phase with the earth's rotation and causes many functions to perform one oscillation per day. Such circadian rhythms (derived from circa dies i.e. about 1 day) were found in almost all classes of plants and animals, and even in protozoans. They persist in a constant environment and, therefore, are independent of any known external trigger signals. Since even unicells perform circadian rhythms which are similar to those observed in highly developed multicellular organisms many scientists favor the existence of a basic mechanism common to all kinds of biological clocks that is located somewhere in the single cell and probably comprises many different biochemical reactions. One purpose of this topical meeting was to discuss how organisms respond to the absence of gravity and terrestrial zeitgeber and how they may react to the imposing of hypergravity fields. Another aim was to develop model-mechanisms appropriate to describe these responses.