The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.     

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.     

Crustaceans as a model for microgravity-induced muscle atrophy.

Atrophy of skeletal muscles is a serious problem in a microgravity environment. It is hypothesized that the unloading of postural muscles, which no longer must resist gravity force, causes an accelerated breakdown of contractile proteins, resulting in a reduction in muscle mass and strength. A crustacean model using the land crab, Gecarcinus lateralis, to assess the effects of spaceflight on protein metabolism is presented. The model is compared to a developmentally-regulated atrophy in which a premolt reduction in muscle mass allows the withdrawal of the large claws at molt. The biochemical mechanisms underlying protein breakdown involves both Ca(2+)-dependent and multicatalytic proteolytic enzymes. Crustacean claw muscle can be used to determine the interactions between shortening and unloading at the molecular level.     

Changes in plant medium composition after a spaceflight experiment: Potassium levels are of special interest

We present results on the analysis of 100 mL medium samples extracted from sterilized foam (Smithers-Oasis, Kent OH) used to support the growth of a representative dicotyledon (Haplopappus gracilis) and a representative monocotyledon (Hemerocallis cv Autumn Blaze) in NASA’s Plant Growth Unit (PGU) during a 5-day Space Shuttle flight and ground experiments. At recovery, the media remaining within replicate (n = 5) foam blocks (for both the spaceflight and ground experiments) were extracted under vacuum, filtered and subjected to elemental analyses. A unique aspect of this experiment was that all plants were either aseptically-generated tissue culture propagated plantlets or aseptic seedling clones. The design of the PGU facilitated the maintenance of asepsis throughout the mission (confirmed by post-flight microbial sampling) and thus any possible impact of microorganisms on medium composition was eliminated. Concentration levels of some elements remained the same, while some decreased and others increased. There was a significant two-fold difference between the final concentrations of potassium when the Earth-based and microgravity experiments were contrasted.     

Evaluation of upper body muscle activity during cardiopulmonary resuscitation performance in simulated microgravity

Performance of efficient single-person cardiopulmonary resuscitation (CPR) is vital to maintain cardiac and cerebral perfusion during the 2–4 min it takes for deployment of advanced life support during a space mission. The aim of the present study was to investigate potential differences in upper body muscle activity during CPR performance at terrestrial gravity (+1Gz) and in simulated microgravity (μG). Muscle activity of the triceps brachii, erector spinae, rectus abdominis and pectoralis major was measured via superficial electromyography in 20 healthy male volunteers. Four sets of 30 external chest compressions (ECCs) were performed on a mannequin. Microgravity was simulated using a body suspension device and harness; the Evetts–Russomano (ER) method was adopted for CPR performance in simulated microgravity. Heart rate and perceived exertion via Borg scores were also measured. While a significantly lower depth of ECCs was observed in simulated microgravity, compared with +1Gz, it was still within the target range of 40–50 mm. There was a 7.7% decrease of the mean (±SEM) ECC depth from 48 ± 0.3 mm at +1Gz, to 44.3 ± 0.5 mm during microgravity simulation (p < 0.001). No significant difference in number or rate of compressions was found between the two conditions. Heart rate displayed a significantly larger increase during CPR in simulated microgravity than at +1Gz, the former presenting a mean (±SEM) of 23.6 ± 2.91 bpm and the latter, 76.6 ± 3.8 bpm (p < 0.001). Borg scores were 70% higher post-microgravity compressions (17 ± 1) than post +1Gz compressions (10 ± 1) (p < 0.001). Intermuscular comparisons showed the triceps brachii to have significantly lower muscle activity than each of the other three tested muscles, in both +1Gz and microgravity. As shown by greater Borg scores and heart rate increases, CPR performance in simulated microgravity is more fatiguing than at +1Gz. Nevertheless, no significant difference in muscle activity between conditions was found, a result that is favourable for astronauts, given the inevitable muscular and cardiovascular deconditioning that occurs during space travel.     

Effects of microgravity on muscle and cerebral cortex: a suggested interaction.

The "slow" antigravity muscle adductor longus was studied in rats after 14 days of spaceflight (SF). The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light and electron microscopy revealed myofiber atrophy, segmental necrosis and regenerative myofibers. Regenerative myofibers were N-CAM immunoreactive (N-CAM-IR). The neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles, degenerative changes, vacant axonal spaces and changes suggestive of axonal sprouting. No alterations of muscle spindles was seen either by light or electron microscopy. These observations suggest that muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight. In a separate study, GABA immunoreactivity (GABA-IR) was evaluated at the level of the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension ("simulated" microgravity). A reduction in number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-IR terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system after spaceflight and hindlimb suspension it is suggested that after limb unloading there are alterations of afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the changes observed in GABA immunoreactivity of cells and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters evaluated in this study provide the basic ground work and pre-flight assessment needed to justify a model for microgravity studies with jatropha in vitro cell cultures. Future studies should focus on results of experiments performed with jatropha in vitro cultures in microgravity.     

Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% (p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO–EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.     

Pituitary cells in space.

Cells of the mammalian pituitary gland synthesize and secrete several protein hormones which regulate a number of organ systems throughout the body. These include the musculoskeletal, immune, vascular and endocrine systems. Since changes occur in these tissues as a result of spaceflight, and since pituitary growth hormone (GH) and prolactin (PRL) play a role in the control of these systems on earth, we have focused attention over the last 10 years on GH and PRL cell function during and after spaceflight. The cumulative results of 4 spaceflight missions and several mimicked microgravity experiments establish 1) that production and release of biologically active GH and PRL is repeatedly and significantly attenuated (usually > 50%) and 2) that changes in cell morphology also occur. In this paper we describe our results within the framework of methodologies and approaches frequently used to study pituitary cell function on earth. In so doing we hope to develop future flight experiments aimed at uncovering possible microgravity "sensing systems" within the pituitary cell.     

Review of primary spaceflight-induced and secondary reloading-induced changes in slow antigravity muscles of rats.

We have examined the light and electron microscopic properties of hindlimb muscles of rats flown in space for 1-2 weeks on Cosmos biosatellite flights 1887 and 2044 and Space Shuttle missions Spacelab-3, Spacelab Life Sciences-1 and Spacelab Life Sciences-2. Tissues were obtained both inflight and postflight permitting definition of primary microgravity-induced changes and secondary reentry and gravity reloading-induced alterations. Spaceflight causes atrophy and expression of fast fiber characteristics in slow antigravity muscles. The stresses of reentry and reloading reveal that atrophic muscles show increased susceptibility to interstitial edema and ischemic-anoxic necrosis as well as muscle fiber tearing with disruption of contractile proteins. These results demonstrate that the effects of spaceflight on skeletal muscle are multifaceted, and major changes occur both inflight and following return to Earth's gravity.     

Involvement of nitric oxide in the mechanism of biochemical alterations induced by simulated microgravity in Microcystis aeruginosa

Simulated microgravity (SMG) can inhibit proliferation and enhance microcystin production of Microcystis aeruginosa. We investigated the role of nitric oxide (NO) in regulating the SMG induced changes of proliferation, photochemical system II photochemical activity, pigment, soluble protein and microcystin production in M. aeruginosa. M. aeruginosa was exposed to 0.1 mM sodium nitroprusside (SNP, NO donor) or 0.02 mM 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, NO scavenger) alone or in combination with SMG for 48 h. SMG and SNP inhibited the growth of M. aeruginosa while c-PTIO had no effect on cell number. As to yield, the negative effect of SMG was augmented by SNP and suppressed by c-PTIO. The intracellular concentrations of chlorophyll a, carotenoid, phycocyanin, soluble protein and microcystin were increased by SMG after 48 h. The effects of SMG on these metabolic processes could be enhanced by SNP and be partly eliminated by c-PTIO. Moreover, SNP and c-PTIO only functioned in these biochemical processes under SMG, unlike in the regulation of cell proliferation and yield. These results showed that the effects of SMG could be enhanced by adding exogenous NO and be mitigated by scavenging endogenous NO, revealing the involvement of NO in the changes in biochemistry processes induced by SMG in M. aeruginosa.     

One dimensional blood flow in a planetocentric orbit

All life on earth is accustomed to the presence of gravity. When gravity is altered, biological processes can go awry. It is of great importance to ensure safety during a spaceflight. Long term exposure to microgravity can trigger detrimental physiological responses in the human body. Fluid redistribution coupled with fluid loss is one of the effects. In particular, in microgravity blood volume is shifted towards the thorax and head. Sympathetic nervous system-induced vasoconstriction is needed to maintain arterial pressure, while venoconstriction limits venous pooling of blood prevents further reductions in venous return of blood to the heart. In this paper, we modify an existing one dimensional blood flow model with the inclusion of the hydrostatic pressure gradient that further depends on the gravitational field modified by the oblateness and rotation of the Earth. We find that the velocity of the blood flow V_B is inversely proportional to the blood specific volume d, also proportional to the oblateness harmonic coefficient J₂, the angular velocity of the Earth ω_E, and finally proportional to an arbitrary constant c. For c = −0.39073 and ξ_H = −0.5 mmHg, all orbits result to less blood flow velocities than that calculated on the surface of the Earth. From all considered orbits, elliptical polar orbit of eccentricity e = 0.2 exhibit the largest flow velocity V_B = 1.031 m/s, followed by the orbits of inclination i = 45°and 0°. The Earth’s oblateness and its rotation contribute a 0.7% difference to the blood flow velocity.     

Comparison of the effects of spaceflight and hindlimb-suspension on rat pituitary vasopressin and brainstem norepinephrine content.

To compare actual spaceflight to ground-based simulation (hindlimb-suspension), we measured the norepinephrine (NE) content in A1, A2, A5 and A6 (locus coeruleus) and the vasopressin content in the neurohypophysial system. The experimental period was of 9 days' duration. The NE content in the locus coeruleus decreased significantly in rats flown for 9 days (67%, p < 0.001), but showed no significant changes after hindlimb-suspension. These results demonstrated that suspended rats adapted better to weightlessness-simulation than flown rats to actual microgravity. In rats flown aboard SLS-1, the vasopressin content was significantly increased in the posterior pituitary (71%, p < 0.01), and was decreased in the hypothalamus (49%, p < 0.05). In 9-day suspended rats pituitary vasopressin levels were unchanged, while in the hypothalamus a significant decrease was noted (21%, p < 0.05). It was concluded that spaceflight changes in pituitary vasopressin levels and in the locus coeruleus NE content were consistent with a stress reaction, occurring during and/or after landing. These results confirmed that hindlimb-suspension model constitutes a valid and less stressful [correction of lesstressful] ground-based simulation of microgravity in rats.     

Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.     

Alterations in glucose and protein metabolism in animals subjected to simulated microgravity.

Reduction of physical activity due to disease or environmental restraints, such as total bed rest or exposure to spaceflight, leads to atrophy of skeletal muscle and is frequently accompanied by alterations in food intake and the concentration of metabolic regulatory hormones such as insulin. Hindlimb suspension of laboratory rats, as a model for microgravity, also shows marked atrophy of gravity dependent muscles along with a reduced gain in body weight. Suspended rats exhibit enhanced sensitivity to insulin-induced glucose uptake when compared with normal control rats and resistance to insulin action when compared with control rats matched similarly for reduced body weight gain. These changes are accompanied by decreased insulin binding and tyrosine kinase activity in soleus but not plantaris muscle, unchanged glucose uptake by perfused hindlimb and decreased sensitivity but not responsiveness to insulin-induced suppression of net proteolysis in hindlimb skeletal muscle. These findings suggest that loss of insulin sensitivity during muscle atrophy is associated with decreased insulin binding and tyrosine kinase activity in atrophied soleus muscle along with decreased sensitivity to the effects of insulin on suppressing net protein breakdown but not on enhancing glucose uptake by perfused hindlimb.