Gravity, cellular membranes and associated processes: an introduction.

The aim of the session "Gravity: Cellular Membranes and Associated Processes" was to bring together scientists from different biological disciplines concentrated on the same scientific question: What are the basic interactions or influences, respectively, of gravity on cellular, molecular level? Presentations were selected dealing with the interaction of gravity with basic physico-chemical processes of membranes, such as changes of the membrane composition of human erythrocytes up to ultrastructural changes in a fungus and other objects after exposure to the conditions of space flight, clinorotation or increased acceleration by means of centrifugation. Taken together the data presented here and in the other session of the symposium on "Life and Gravity", clearly outline that future basic physico-chemical studies, and studies dealing with the molecular basis of the cellular signal-perception and transduction, have to be pressed forward in order to understand signal-responses on cellular level, but also of a whole organism.     

Physical effects at the cellular level under altered gravity conditions.

Several modifications of differentiated functions of animal cells cultivated in vitro have been reported when cultures have been exposed to increased or decreased inertial acceleration fields by centrifugation, clinorotation, and orbital space flight. Variables modified by clinorotation conditions include inertial acceleration, convection, hydrostatic pressure, sedimentation, and shear stress, which also affect transport processes in the extracellular chemical environment. Autocrine, paracrine and endocrine substances, to which cells are responsive via specific receptors, are usually transported in vitro (and possibly in certain embryos) by convection and in vivo by a circulatory system or ciliary action. Increased inertial acceleration increases convective flow, while microgravity nearly abolishes it. In the latter case the extracellular transport of macromolecules is governed by diffusion. By making certain assumptions it is possible to calculate the Peclet number, the ratio of convective transport to diffusive transport. Some, but not all, responses of cells in vitro to modified inertial environments could be manifestations of modified extracellular convective flow.     

The SOS-LUX-LAC-FLUORO-Toxicity-test on the International Space Station (ISS).

In the 21st century, an increasing number of astronauts will visit the International Space Station (ISS) for prolonged times. Therefore it is of utmost importance to provide necessary basic knowledge concerning risks to their health and their ability to work on the station and during extravehicular activities (EVA) in free space. It is the aim of one experiment of the German project TRIPLE-LUX (to be flown on the ISS) to provide an estimation of health risk resulting from exposure of the astronauts to the radiation in space inside the station as well as during extravehicular activities on one hand, and of exposure of astronauts to unavoidable or as yet unknown ISS-environmental genotoxic substances on the other. The project will (i) provide increased knowledge of the biological action of space radiation and enzymatic repair of DNA damage, (ii) uncover cellular mechanisms of synergistic interaction of microgravity and space radiation and (iii) examine the space craft milieu with highly specific biosensors. For these investigations, the bacterial biosensor SOS-LUX-LAC-FLUORO-Toxicity-test will be used, combining the SOS-LUX-Test invented at DLR Germany (Patent) with the commercially available LAC-FLUORO-Test. The SOS-LUX-Test comprises genetically modified bacteria transformed with the pBR322-derived plasmid pPLS-1. This plasmid carries the promoterless lux operon of Photobacterium leiognathi as a reporter element under control of the DNA-damage dependent SOS promoter of ColD as sensor element. This system reacts to radiation and other agents that induce DNA damages with a dose dependent measurable emission of bioluminescence of the transformed bacteria. The analogous LAC-FLUORO-Test has been developed for the detection of cellular responses to cytotoxins. It is based on the constitutive expression of green fluorescent protein (GFP) mediated by the bacterial protein expression vector pGFPuv (Clontech, Palo Alto, USA). In response to cytotoxic agents, this system reacts with a dose-dependent reduction of GFP-fluorescence. Currently, a fully automated miniaturized hardware system for the bacterial set up, which includes measurements of luminescence and fluorescence or absorption and the image analysis based evaluation is under development. During the first mission of the SOS-LUX-LAC-FLUORO-Toxicity-Test on the ISS, a standardized, DNA-damaging radiation source still to be determined will be used as a genotoxic inducer. A panel of recombinant Salmonella typhimurium strains carrying either the SOS-LUX plasmid or the fluorescence-mediating lac-GFPuv plasmid will be used to determine in parallel on one microplate the genotoxic and the cytotoxic action of the applied radiation in combination with microgravity. Either in addition to or in place of the fluorometric measurements of the cytotoxic agents, photometric measurements will simultaneously monitor cell growth, giving additional data on survival of the cells. The obtained data will be available on line during the TRIPLE-LUX mission time. Though it is the main goal during the TRIPLE-LUX mission to measure the radiation effect in microgravity, the SOS-LUX-LAC-FLUORO-Toxicity-test in principle is also applicable as a biomonitor for the detection and measurement of genotoxic substances in air or in the (recycled) water system on the ISS or on earth in general.     

Experimental and theoretical analysis of the influence of gravity at the cellular level: a review.

The present paper is a review of the experimental investigations published in the literature and performed by the authors on space vehicles. The paper also gives an analysis of theoretical concepts concerning gravitational effects on the cell. Taking this into account, the authors put forth a hypothesis that free-living unicellular organisms are indifferent to variations in the magnitude and direction of the gravitational field.     

Influence of clinorotation and fettering stress on tail regeneration of Triturus vulgaris (Urodela).

Tail-amputated adult Triturus vulgaris, fettered in cuvettes of a fast-rotating clinostat were exposed to simulated weightlessness (60 rpm; equiv. to 10(-3)-10(-4) g), during a 14-day period. To feed and clean the animals rotation was stopped once a day for approx. 10 min. To test the influence of the fettering stress, a second series of animals was kept separately under normal earth conditions without rotation. A further control series was kept in a dark container without any handicap. While tail regeneration of the rotated animals was markedly accelerated, the fettered-only animals showed a considerably less marked acceleration effect. At the end of the 14-day period, all regenerates were reamputated together with an additional 5 mm of the tail stump. Although this second level of amputation was distant from the first, the regenerative growth rate of the rotated series was accelerated 123% in contrast to both the control and the fettered-only series. Our results demonstrate that the growth acceleration is induced by clinorotation. Fettering stress has no comparable influence. The growth promoting effect is not limited to the regenerating area.     

Plant-module for autonomous space support (P-MASS).

A wide variety of technical and science questions arise when attempting to envision the long-term support of plants, algae and bacteria in space. Currently, spaceflight data remain elusive since there are no U.S. carriers for investigating either the germane technical or scientific issues. The first flight of the Commercial Experiment Transporter (COMET) will provide a nominal 30 day orbital opportunity to evaluate such issues. The P-MASS is a small payload that is designed to meet the mass (40 lbs.), volume (1.5 cu.ft.), and power (120 W) constraints of one of several COMET payloads while enabling flight evaluations of plants, algae and bacteria. Various P-MASS subsystems have been subjected to extensive ground tests as well as KCl35 tests. Various biological sub-systems have been similarly evaluated. Through a variety of sensors coupled with color video, the P-MASS performance and the supported biological systems will be compared for terrestrial controls versus spaceflight materials. This small, low cost payload should return valuable information regarding the requirements for hardware and biological systems needed to move toward bioregenerative life support systems in space. In addition, it should be possible to accurately identify major unresolved difficulties that may arise in the long-term, spaceflight support of various biological systems. Finally, this generic spaceflight capability should enable a variety of plant research programs focused on the use of microgravity to modulate and exploit plant products for commercial applications ranging from new agricultural products to pharmacological feedstocks and new controlled agricultural strategies.     

Life on Mars: how it disappeared (if it was ever there).

The cryptoendolithic microbial community in the Ross Desert (McMurdo Dry Valleys) of Antarctica exists at temperatures significantly below the temperature optima of the primary producers. Surviving near the limit of their physiological adaptability, the organisms are under severe environmental stress, so further deterioration in the environment results in cell damage and death. The sequence of events leading to extinction is considered to be a terrestrial analog for disappearance of possible life on early Mars. Progressive stages of cell damage and death in the Ross Desert material are documented with transmission electron microscopy.     

Water wave communication in the genus Bombina (amphibia).

Amphibians were phylogenetically the first vertebrates to leave the aquatic environment and cope with terrestrial conditions including effects of gravity and substrate on movement and communication. Studies of extant primitive amphibians, which have conserved ancestral morphology and behavior, may help us to understand how gravitational adaptation from aquatic to terrestrial environments occurred. The anuran genus Bombina is a candidate for this type of investigation. In particular, a member of this genus, B. orientalis, is known for its low reaction threshold to minor changes of angular acceleration. We hypothesize that a heightened sensitivity to angular and mechanical accelerations evolved with wave communication. Comparisons of such behavior among B. variegata, B. bombina and B. orientalis may shed light on the evolution of reproductive systems based on water wave communication and relevant vestibular sensitivity. This may represent a transition to derived vocalization modes, which is seen in B. bombina to a certain degree.     

Free flow electrophoresis in space shuttle program (Biotex).

In the space shuttle program free flow electrophoresis will be applied for separation of proteins, biopolymers and cells. Proteins are to be separated according to the "Feldsprung-Gradienten" procedure by Prof. H. Wagner, University of Saarbruecken, biopolymers are to be separated by the isotachophoresis technique by Prof. Schmitz, University of Muenster and we intend to separate cells in order to increase the efficiency of recovery of hybrid cells after electrofusion performed under microgravity in collaboration with Prof. U. Zimmermann, University of Wuerzburg. There are supposed two ways for reaching this goal: 1) Enrichment of cells before electrofusion may enhance the probability that the cells of interest are immortalized. 2) Separation of cells after electrofusion may help to clone the hybrid cells of interest. Under microgravity, the combination of improved electrophoresis with higher electrofusion rates may provide new possibilities for immortalization of cells. This may be a new way to obtain cellular products, which are physiologically glycosylated.     

Differentiation in microgravity of neural and muscle cells of a vertebrate (amphibian).

The CELIMENE space experiment (CELulles en Impesanteur: Muscle Et Neurone Embryonnaires) was devoted to the study of the influence of gravity on the differentiation, the organisation and the maintenance of the highly specialised nervous system and muscular system. CELIMENE was carried out during the first flight of the IBIS hardware (Instrument for BIology in Space) with the fully automatic space mission PHOTON 10 in February 1995. Using the amphibian Pleurodeles waltl as a vertebrate model, in vitro experiments involved immunocytochemical detection of glial-, neuronal- and muscle-specific markers, and neurotransmitters in cells developed under conditions of microgravity compared with 1g controls, on-board and on the ground. We observed that the altered gravity did not disturb cell morphogenesis or differentiation.     

Overview of the space environmental effects observed on the retrieved Long Duration Exposure Facility (LDEF).

The Long Duration Exposure Facility (LDEF), which encompassed 57 experiments with more than 10,000 test specimens, spent 69 months in low Earth orbit (LEO) before it was retrieved by the Space Shuttle in January 1990. Hundreds of LDEF investigators, after studying for over two years these retrieved test specimens and the onboard recorded data and systems hardware, have generated a unique first-hand view of the long term synergistic effects that the LEO environment can have on spacecraft. These studies have also contributed significantly toward more accurate models of the LEO radiation, meteoroid, manmade debris and atomic oxygen environments. This paper provides an overview of some of the many LDEF observations and the implications these can have on future spacecraft such as Space Station Freedom.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

Possible actions of gravity on the cellular machinery.

Since the first flight of the ESA Biorack on the German Spacelab Mission D1 in 1985 evidence has been obtained that biological cells and small unicellular organisms function differently under conditions of microgravity. However, there is still lack of scientific proof that these effects are caused by a direct influence on the cells in the weightlessness condition. The question how normal gravity may play a role in cellular activity is being addressed and the results show that gravity may provide important signals during certain state transitions in the cell. These would be gravity-sensitive windows in the biological process. Also, by amplification mechanisms inside the cell, the cell may assume a state that is typical for normal gravity conditions and would change in microgravity. Experimental tools are discussed that would provide the conditions to obtain evidence for direct action of gravity and for the possible existence of gravity-sensitive windows.     

Swimming behavior of Paramecium--first results with the low-speed centrifuge microscope (NIZEMI).

The low-speed centrifuge microscope NIZEMI (= Nieder-Geschwindigkeits-Zentrifugen-Mikroskop) is an excellent tool with which to investigate the effects of slightly increased gravity in the fields of biology and material sciences. We investigated the swimming behavior of Paramecium in the NIZEMI, by aid of a computer-controlled image analysis system. In the range of acceleration (1 g to 5 g), cells retained their swimming capability, did not sediment, and even increased the precision of their negative gravitaxis but reduced their mean swimming velocity.     

Utilization of sweet potatoes in controlled ecological life support systems (CELSS).

A number of studies have selected the sweet potato as a potentially important crop for CELSS. Most hydroponic studies of sweet potatoes have been short term (<80 days). Full term (90 to 150 days) studies of sweet potatoes in hydroponic systems were needed to understand the physiology of storage root enlargement and to evaluate sweet potato production potential for CELSS. Early and late maturing sweet potato varieties were crown in hydroponic systems of different types--static with periodic replacement, flowing with and without recirculation, aggregate, and non-aggregate. In a flowing system with recirculation designed at Tuskegee University using the nutrient film technique (NFT), storage root yields as high as 1790 g were produced with an edible growth rate of up to 66 g m-2 d-1 and a harvest index as high as 89% under greenhouse conditions. Preliminary experiments indicated high yields can be obtained in controlled environmental chambers. Significant cultivar differences were found in all systems studied. Nutritive composition of storage roots and foliage were similar to field-grown plants. The results indicate great potential for sweet potato in CELSS.     

Prostaglandin-induced radioprotection of murine intestinal crypts and villi by a PGE diene analog (SC-44932) and a PGI analog (iloprost).

The aminothiols exemplified by WR-2721 are effective radioprotectors; however, their toxicity associated with hypotension, nausea, and emesis has limited their development for applications to medicine or in harzardous radiation environments. There is a need for new radioprotectors that have fewer toxic side effects when given alone or combined with reduced amounts of thiols. A variety of prostaglandins (PGs) have been shown to be radioprotective agents and some appear to have fewer toxic side effects than the aminothiols. Iloprost, a stable PGI, analog protects the clonogenic epithelial cells of intestinal crypts but does not protect epithelial cells of the villi. In contrast, an E-series omega chain diene analog designated SC-44932 protects epithelial cells of both crypts and villi. When the two are combined, protection of the crypts is additive and the villi are protected to the same degree as when SC-44932 is given alone. Since radioprotection for some PGs has been shown to be dependent upon receptors, we suggest that the pattern of radioprotection seen with these two analogs depend on the location of the respective receptors or on the ability of differentiated villus cells to respond to PGs. By studying different analogs, we hope to identify mechanisms associated with PG-induced radioprotection and to identify the most protective PG analogs for applications of radioprotection.     

Calcium movements and the cellular basis of gravitropism.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.     

The effect of geocenter motion on Jason-2 orbits and the mean sea level

We compute a series of Jason-2 GPS and SLR/DORIS-based orbits using ITRF2005 and the std0905 standards ( Lemoine et al., 2010). Our GPS and SLR/DORIS orbit data sets span a period of 2 years from cycle 3 (July 2008) to cycle 74 (July 2010). We extract the Jason-2 orbit frame translational parameters per cycle by the means of a Helmert transformation between a set of reference orbits and a set of test orbits. We compare the annual terms of these time-series to the annual terms of two different geocenter motion models where biases and trends have been removed. Subsequently, we include the annual terms of the modeled geocenter motion as a degree-1 loading displacement correction to the GPS and SLR/DORIS tracking network of the POD process. Although the annual geocenter motion correction would reflect a stationary signal in time, under ideal conditions, the whole geocenter motion is a non-stationary process that includes secular trends. Our results suggest that our GSFC Jason-2 GPS-based orbits are closely tied to the center of mass (CM) of the Earth consistent with our current force modeling, whereas GSFC’s SLR/DORIS-based orbits are tied to the origin of ITRF2005, which is the center of figure (CF) for sub-secular scales. We quantify the GPS and SLR/DORIS orbit centering and how this impacts the orbit radial error over the globe, which is assimilated into mean sea level (MSL) error, from the omission of the annual term of the geocenter correction. We find that for the SLR/DORIS std0905 orbits, currently used by the oceanographic community, only the negligence of the annual term of the geocenter motion correction results in a – 4.67 ± 3.40 mm error in the Z-component of the orbit frame which creates 1.06 ± 2.66 mm of systematic error in the MSL estimates, mainly due to the uneven distribution of the oceans between the North and South hemisphere.