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NASA NTRS1997

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

NASA Program Space Physiology and Countermeasures↗

NASA NTRS1996

Effects of altered loading states on muscle plasticity: what have we learned from rodents?

This paper summarizes the key findings concerning the adaptive properties of rodent muscle in response to altered loading states. When the mechanical stress on the muscle is chronically increased, the muscle adapts by hypertrophying its fibers. This response is regulated by processes resulting in contractile protein expression reflecting slower phenotypes, thereby enabling the muscle to better support load-hearing activity. In contrast, reducing the load-bearing activity induces an opposite response whereby muscles used for both antigravity function and locomotion atrophy while transforming some of the slow fibers into faster contractile phenotypes. Accompanying the atrophy is both a reduced power generating and activity sustaining capability. These adaptive processes are regulated by both transcriptional and translational processes. Available evidence further suggests that the interaction of heavy resistance activity and hormonal/growth factors (insulin-like growth factor, growth hormone, glucocorticoids, etc.) are critical in the maintenance of muscle mass and function. Also resistance training, in contrast to other activities such as endurance running, provides a more economical form of stress because less mechanical activity is required to maintain muscle homeostasis in the context of chronic states of weightlessness.

Non-NASA Center↗

NASA NTRS1996

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

NASA Discipline Radiation Health↗

NASA NTRS1994

Experiment K-7-16: Effects of Microgravity or Simulated Launch on Testicular Function in Rats

Fixed or frozen testicular tissues from five rats per group were analyzed by: subjective and quantitative evaluations of spermatogenesis; Northern-blot analysis for expression of selected genes; quantification of testosterone and receptors for LH; and morphometric analysis of Leydig cells. Based on observations of fixed tissue, it was evident that some rats in the flight and vivarium groups had testicular abnormalities unassociated with treatment, and probably existing when they were assigned randomly to the four treatment groups; the simulated-launch group contained no abnormal rat. Lesions induced in testes of caudal-elevation rats precluded discernment of any pre-existing abnormality. Considering rats without pre-existing abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (E less than 0.05) in flight rats than in simulated-launch or vivarium rats. However, ratios of germ cells to each other, or to Sertoli cells, and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. There was no effect of flight on normal expression of testis-specific hsp gene products, or evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rLH in testicular tissue, and surface densities of smooth endoplasmic reticulum and peroxisomes in Leydig cells, were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20 percent of values for simulated-launch or vivarium controls. Thus, spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Sequela of reduced androgen production on turnover of muscle and bone should be considered when interpreting data from mammals exposed to microgravity.

Amann, R. P.↗

NASA NTRSDate not supplied

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

Maria Moreno-Villanueva↗

NASA NTRS2024

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

María Moreno-Villanueva↗

NASA NTRS2019

Prenatal 2g Exposure Alters Placental Expression of Stress-Related Genes

Extra-terrestrial colonization is of growing interest to space agencies and private entities, emphasizing the importance of research on reproduction and development in the absence of Earth's 1G. Maternal stressors can modify offspring development, exerting significant lifespan and crossgenerational changes through prenatal programming. The space environment is stressful, therefore exposure to altered gravity during pregnancy may impact later life outcomes in offspring. In ground-based studies, we exposed pregnant rats to continuous +G (above Earth gravity), and observed overweight and elevated anxiety in adult male (but not female) offspring, common phenotypes associated with prenatal maternal stress. Here we hypothesize that exposure to increased gravity during pregnancy elicits changes in the expression of stress-related genes in placenta that may mediate emergence of later life outcomes. While the placenta transports maternal factors to the fetus and produces endogenous fetal hormones, stress-induced changes at the placental-uterine interface may also alter communication between mother and fetus, facilitating prenatal transmission of unfavorable later life outcomes and cross-generational epigenetic alterations. Maternal stress elevates maternal glucocorticoids however placental 11b-hydroxysteroid dehydrogenase type 2 (HSD11B2) buffers fetal exposure by converting cortisol/corticosterone into inactive metabolites. Maternal stress during pregnancy down-regulates this enzyme and can induce epigenetic changes in placental and fetal tissues accounting for heightened adult HPA reactivity. Past studies have shown a placenta-specific increase in DNA methyltransferase (DNMT3a) mRNA in stressed mothers, an effect with implications for genome-wide epigenetic changes that may account for diverse phenotypic outcomes following maternal stress. Here we exposed groups of pregnant rats to one of five gravity loads (1, 1.5, 1.75 and 2G) and analyzed placental samples during late gestation. We predicted a systematic dose-response relationship between gravity load and the expression of the HSD11B2 and DNMT3 genes, thereby linking maternal exposure to altered gravity during pregancy with maternal stress.

Kalotia, Simranjit K.↗

NASA NTRSDate not supplied

Differential Responses to Mechanostimulation in Embryonic Stem Cells Versus the Embryoid Body Model of Development Assessed at Single Cell RNA-Seq Resolution

Mechanicalforces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. In contrast disuse canreduce normal mechanical loading, resulting in altered cell and tissue function. Although loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how cells respondto this stimulusduring early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-minute pulse of 50xg hypergravity-generated 5kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the Embryoid Body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the hydrostatic pressurepulse. Single cell tSNE mapping shows limited transcriptome shifts in response to thispulse in either ESCs or EBs; this pulse,however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program.We assessed ESCs and EBs for differentially expressed (DE) genes with hydrostatic pressurepulse and found approximately 1/3 DE genes were shared. However, gene ontology (GO) pathway analysis show that EBs have choreographed responses associated with upregulation ofpathways formulticellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs showsmechanostimulationpromotes maintenance of transitory cell phenotypes in early development,including EB cluster co-expression of markers for progenitor, post-implant epiblast and primitive endoderm phenotypes versus expression exclusivity in the non-pulsed clusters. Pseudotime analysisidentified three branching cell types susceptible tohydrostatic pressureinduction of cell fate decisions. In summary, this study provides novel evidence that ESC maintenance and EB development can be regulated by mechanostimulation,and that stem cells committed to a differentiation program are more sensitive to force-induced changes to their transcriptome.

Cassandra Juran↗

NASA NTRS1991

Space charge enhanced plasma gradient effects on satellite electric field measurements

It has been recognized that plasma gradients can cause error in magnetospheric electric field measurements made by double probes. Space charge enhanced Plasma Gradient Induced Error (PGIE) is discussed in general terms, presenting the results of a laboratory experiment designed to demonstrate this error, and deriving a simple expression that quantifies this error. Experimental conditions were not identical to magnetospheric conditions, although efforts were made to insure the relevant physics applied to both cases. The experimental data demonstrate some of the possible errors in electric field measurements made by strongly emitting probes due to space charge effects in the presence of plasma gradients. Probe errors in space and laboratory conditions are discussed, as well as experimental error. In the final section, theoretical aspects are examined and an expression is derived for the maximum steady state space charge enhanced PGIE taken by two identical current biased probes.

Diebold, Dan↗

NASA NTRS1992

The dynamics of rain-induced fades

The dynamics of rain-induced fades on satellite radio links is studied by evaluating the rate at which the first Fresnel zone volume fills with rain. A compact expression for the fade slope on a terrestrial path is derived which shows that once the rain rate is specified, fade slope is very sensitive to differences in rain velocity. Thus, there is no unique relationship between fade slope and rain rate.

Sweeney, Dennis G.↗

NASA NTRS1993

A constitutive model for the forces of a magnetic bearing including eddy currents

A multiple magnet bearing can be developed from N individual electromagnets. The constitutive relationships for a single magnet in such a bearing is presented. Analytical expressions are developed for a magnet with poles arranged circumferencially. Maxwell's field equations are used so the model easily includes the effects of induced eddy currents due to the rotation of the journal. Eddy currents must be included in any dynamic model because they are the only speed dependent parameter and may lead to a critical speed for the bearing. The model is applicable to bearings using attraction or repulsion.

Taylor, D. L.↗

NASA NTRS1999

PKC Isoform Expression in Modeled Microgravity

Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

Risin, Diana↗

NASA NTRS1988

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21 degrees C. Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R. Using this technique we show for the first time that Ca2+ contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea.

NASA Discipline Plant Biology↗

NASA NTRS1993

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Non-NASA Center↗

NASA NTRSDate not supplied

Life Finds A Way, the Dynamics of E. Coli Evolution in Microgravity

Investigating the evolutionary dynamics of Escherichia coli in microgravity offers a unique opportunity to understand microbial adaptation to extreme environments. Here, we explored the effects of simulated microgravity (SµG) on gene expression and genome evolution of Escherichia coli REL606, a strain continuously evolved and documented terrestrially for 35 years. We used transcriptomic profiling over a 24-hour growth cycle to examine how short-term exposure to SµG under glucose-limiting and glucose-replete conditions may influence the genetic adaptations in microbial populations. Pathway analyses of differentially expressed genes suggest that SµG may alter cell membrane structure and function across all conditions, while changes to protein synthesis machinery were uniquely observed in glucose-replete samples. Furthermore, altered expression of several prophage genes across conditions in SµG samples and upregulation of general stress response factors hints at the potential for stress-induced mutagenesis in response to microgravity. We further investigated the impact of long-term exposure to SµG on genome evolution and observed a more rapid accumulation of base substitutions and deletions in SµG sample genomes across time. Specifically, mutations in the mraZ and elyC genes suggest a mechanism for increased production of peptidoglycan in the cell membrane. These findings offer insights into bacterial adaptations in long-term microgravity environments and pave the way for further detailed investigations.

Brittney Lozzi↗

NASA NTRS1994

Superfluid helium sloshing dynamics induced oscillations and fluctuations of angular momentum, force and moment actuated on spacecraft driven by gravity gradient or jitter acceleration associated with slew motion

The generalized mathematical formulation of sloshing dynamics for partially filled liquid of cryogenic superfluid helium II in dewar containers driven by the gravity gradient and jitter accelerations associated with slew motion for the purpose to perform scientific observation during the normal spacecraft operation are investigated. An example is given with the Advanced X-Ray Astrophysics Facility-Spectroscopy (AXAF-S) for slew motion which is responsible for the sloshing dynamics. The jitter accelerations include slew motion, spinning motion, atmospheric drag on the spacecraft, spacecraft attitude motions arising from machinery vibrations, thruster firing, pointing control of spacecraft, crew motion, etc. Explicit mathematical expressions to cover these forces acting on the spacecraft fluid systems are derived. The numerical computation of sloshing dynamics is based on the non-inertia frame spacecraft bound coordinate, and solve time-dependent, three-dimensional formulations of partial differential equations subject to initial and boundary conditions. The explicit mathematical expressions of boundary conditions to cover capillary force effect on the liquid-vapor interface in microgravity environments are also derived. The formulations of fluid moment and angular moment fluctuations in fluid profiles induced by the sloshing dynamics, together with fluid stress and moment fluctuations exerted on the spacecraft dewar containers have also been derived. Examples are also given for cases applicable to the AXAF-S spacecraft sloshing dynamics associated with slew motion.

Hung, R. J.↗

NASA NTRS2000

Chronic N(G)-nitro-L-arginine methyl ester-induced hypertension : novel molecular adaptation to systolic load in absence of hypertrophy

BACKGROUND: Chronic N(G)-nitro-L-arginine methyl ester (L-NAME), which inhibits nitric oxide synthesis, causes hypertension and would therefore be expected to induce robust cardiac hypertrophy. However, L-NAME has negative metabolic effects on protein synthesis that suppress the increase in left ventricular (LV) mass in response to sustained pressure overload. In the present study, we used L-NAME-induced hypertension to test the hypothesis that adaptation to pressure overload occurs even when hypertrophy is suppressed. METHODS AND RESULTS: Male rats received L-NAME (50 mg. kg(-1). d(-1)) or no drug for 6 weeks. Rats with L-NAME-induced hypertension had levels of systolic wall stress similar to those of rats with aortic stenosis (85+/-19 versus 92+/-16 kdyne/cm). Rats with aortic stenosis developed a nearly 2-fold increase in LV mass compared with controls. In contrast, in the L-NAME rats, no increase in LV mass (1. 00+/-0.03 versus 1.04+/-0.04 g) or hypertrophy of isolated myocytes occurred (3586+/-129 versus 3756+/-135 microm(2)) compared with controls. Nevertheless, chronic pressure overload was not accompanied by the development of heart failure. LV systolic performance was maintained by mechanisms of concentric remodeling (decrease of in vivo LV chamber dimension relative to wall thickness) and augmented myocardial calcium-dependent contractile reserve associated with preserved expression of alpha- and beta-myosin heavy chain isoforms and sarcoplasmic reticulum Ca(2+) ATPase (SERCA-2). CONCLUSIONS: When the expected compensatory hypertrophic response is suppressed during L-NAME-induced hypertension, severe chronic pressure overload is associated with a successful adaptation to maintain systolic performance; this adaptation depends on both LV remodeling and enhanced contractility in response to calcium.

Non-NASA Center↗

NASA NTRS1988

Star formation via the phase transition of an adiabatic gas

An analytic model based on a second-order accurate global virial analysis is used to describe two stable equilibrium states for rotating gas clouds: a pressure-supported, diffuse state and a rotationally supported, compact state. Diffuse and compact equilibrium sequences are identified along which slow, secular evolution of a gas cloud can occur. A virial analysis provides analytic expressions for the limiting masses of gas clouds at points of dynamical instability. The minimum mass below which external triggers of any amplitude cannot effectively induce star formation is identified for adiabatic gas clouds. It is suggested that fluctuations in the ISM of some galaxies may actually cause star formation to proceed primarily via a phase transition, rather than via the classical Jeans instability.

Tohline, Joel E.↗