Liposome formation in microgravity.

Liposomes are artificial vesicles with a phospholipid bilayer membrane. The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules and can be observed during the removal of detergent from phospholipids dissolved in detergent micelles. As detergent concentration in the mixed micelles decreases, the non-polar tail regions of phospholipids produce a hydrophobic effect that drives the micelles to fuse and form planar bilayers in which phospholipids orient with tail regions to the center of the bilayer and polar head regions to the external surface. Remaining detergent molecules shield exposed edges of the bilayer sheet from the aqueous environment. Further removal of detergent leads to intramembrane folding and membrane folding and membrane vesiculation, forming liposomes. We have observed that the formation of liposomes is altered in microgravity. Liposomes that were formed at 1-g did not exceed 150 nm in diameter, whereas liposomes that were formed during spaceflight exhibited diameters up to 2000 nm. Using detergent-stabilized planar bilayers, we determined that the stage of liposome formation most influenced by gravity is membrane vesiculation. In addition, we found that small, equipment-induced fluid disturbances increased vesiculation and negated the size-enhancing effects of microgravity. However, these small disturbances had no effect on liposome size at 1-g, likely due to the presence of gravity-induced buoyancy-driven fluid flows (e.g., convection currents). Our results indicate that fluid disturbances, induced by gravity, influence the vesiculation of membranes and limit the diameter of forming liposomes.     

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

Confocal microscopy in microgravity research.

We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.     

Virus protein assembly in microgravity.

The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid. VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure. Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly. Flight studies were conducted on STS-37 on April 5-9, 1991. Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0). After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation. Flight and ground-based samples were analyzed by electron microscopy. Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits. Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.     

Gravitropism of basidiomycetous fungi--on Earth and in microgravity.

In order to achieve perfect positioning of their lamellae for spore dispersal, fruiting bodies of higher fungi rely on the omnipresent force gravity. Only accurate negatively gravitropic orientation of the fruiting body cap will guarantee successful reproduction. A spaceflight experiment during the STS-55 Spacelab mission in 1993 confirmed that the factor gravity is employed for spatial orientation. Most likely every hypha in the transition zone between the stipe and the cap region is capable of sensing gravity. Sensing presumably involves slight sedimentation of nuclei which subsequently causes deformation of the net-like arrangement of F-actin filament strands. Hyphal elongation is probably driven by hormone-controlled activation and redistribution of vesicle traffic and vesicle incorporation into the vacuoles and cell walls to subsequently cause increased water uptake and turgor pressure. Stipe bending is achieved by way of differential growth of the flanks of the upper-most stipe region. After reorientation to a horizontal position, elongation of the upper flank hyphae decreases 40% while elongation of the lower flank slightly increases. On the cellular level gravity-stimulated vesicle accumulation was observed in hyphae of the lower flank.     

Development and growth of potato tubers in microgravity.

A potato explant consisting of a leaf, its axillary bud, and a small segment of stem will develop a tuber in 10-14 days when grown on earth. The tubers develop from the axillary buds and accumulate starch derived from sugars produced through photosynthesis and/or mobilized from leaf tissue. Potato explants were harvested and maintained in the Astroculture (TM) unit, a plant growth chamber designed for spaceflight. The unit provides an environment with controlled temperature, humidity, CO2 level, light intensity, and a nutrient delivery system. The hardware was loaded onto the space shuttle Columbia 24 hours prior to the launch of the STS-73 mission. Explant leaf tissue appeared turgid and green for the first 11 days of flight, but then became chlorotic and eventually necrotic by the end of the mission. The same events occurred to ground control explants with approximately the same timing. At the end of the 16-day mission, tubers were present on each explant. The size and shape of the space-grown tubers were similar to the ground-control tubers. The arrangement of cells in the tuber interior and at the exterior in the periderm was similar in both environments. Starch and protein were present in the tubers grown in space and on the ground. The range in starch grain size was similar in tubers from both environments, but the distribution of grains into size classes differed somewhat, with the space-grown tubers having more small grains than the ground control tubers. Proteinaceous crystals were found in tubers formed in each condition.     

Crystallization of calcium phosphate in microgravity.

Dilute solutions of CaCl2 and KH2PO4 + K2HPO4 were diffusing from either side into a mixing chamber with KCl solution. The microgravity experiment yielded aggregates of large crystals of OCP (Ca8H2(PO4)3,5H2O) and spherolites of smaller, but still visible crystals of HAP (Ca5OH(PO4)3), the stable final phase. Ground-based experiments yielded submicroscopic HAP crystals. Results of calculations of diffusion and crystal growth on the basis of previous knowledge agree well with observations.     

Ultrastructure and cytoskeleton of Chara rhizoids in microgravity.

Gravitropic tip growth of Chara rhizoids is dependent on the presence and functional interaction between statoliths, cytoskeleton and the tip-growth-organizing complex, the Spitzenkorper. Microtubules are essential for the polar cytoplasmic zonation but are excluded from the apex and do not play a crucial role in the primary steps of gravisensing and graviresponse. Actin filaments form a dense meshwork in the subapical zone and converge into a prominent apical actin patch which is associated with the endoplasmic reticulum (ER) aggregate representing the structural center of the Spitzenkorper. The position of the statoliths is regulated by gravity and a counteracting force mediated by actomyosin. Reducing the acceleration forces in microgravity experiments causes a basipetal displacement of the statoliths. Rhizoids grow randomly in all directions. However, they express the same cell shape and cytoplasmic zonation as ground controls. The ultrastructure of the Spitzenkorper, including the aggregation of ER, the assembly of vesicles in the apex, the polar distribution of proplastids, mitochondria, dictyosomes and ER cisternae in the subapical zone is maintained. The unaltered cytoskeletal organization, growth rates and gravitropic responsiveness indicate that microgravity has no major effect on gravitropic tip-growing Chara rhizoids. However, the threshold value of gravisensitivity might be different from ground controls due to the altered position of statoliths, a possibly reduced amount of BaSO4 in statoliths and a possible adaptation of the actin cytoskeleton to microgravity conditions.     

Regulative development of Xenopus laevis in microgravity.

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.     

Long-term effects of microgravity and possible countermeasures.

It is well known that long-term exposure to microgravity causes a number of physiological and biochemical changes in humans; among the most significant are: 1) negative calcium balance resulting in the loss of bone; 2) atrophy of antigravity muscles; 3) fluid shifts and decreased plasma volume; and 4) cardiovascular deconditioning that leads to orthostatic intolerance. It is estimated that a mission to Mars may require up to 300 days in a microgravity environment; in the case of an aborted mission, the astronauts may have to remain in reduced gravity for up to three years. Although the Soviet Union has shown that exercise countermeasures appear to be adequate for exposures of up to one year in space, it is questionable whether astronauts could or should have to maintain such regimes for extremely prolonged missions. Therefore, the NASA Life Sciences Division has initiated a program designed to evaluate a number of methods for providing an artificial gravity environment.     

Ultrastructural changes in osteocytes in microgravity conditions.

We examined the histology and morphometry of biosamples (biopsies) of the iliac crest of monkeys, flown 14 days aboard the "Bion-11", using electron microscopy. We found, that some young osteocytes take part in the activation of collagen protein biosynthesis in the adaptive remodeling process of the bone tissue to microgravity conditions. Osteocyte lacunae filled with collagen fibrils; this correlates with fibrotic osteoblast reorganization in such zones. The osteolytic activity in mature osteocytes is intensified. As a result of osteocyte destruction, the quantity of empty osteocytic lacunae in the bone tissue increases.     

Oxygen requirement of germinating flax seeds.

Plant experiments in earth orbit are typically prepared on the ground and germinated in orbit to study gravity effects on the developing seedlings. Germination requires the breakdown of storage compounds, and this metabolism depends upon respiration, making oxygen one of the limiting factors in seed germination. In microgravity lack of run-off of excess water requires careful testing of water dispensation and oxygen availability. In preparation for a shuttle experiment (MICRO on STS-107) we studied germination and growth of flax (Linum usitatissimum L.) seedlings in the developed hardware (Magnetic Field Chamber, MFC). We tested between four to 32 seeds per chamber (air volume=14 mL) and after 36 h measured the root length. At 90 microliters O2 per seed (32 seeds/chamber), the germination decreased from 94 to 69%, and the root length was reduced by 20%, compared to 8 seeds per chamber. Based on the percent germination and root length obtained in controlled gas mixtures between 3.6 and 21.6% O2 we determined the lower limit of reliable germination to be 10 vol. % O2 at atmospheric pressure. Although the oxygen available in the MFC's can support the intended number of seeds, the data show that seed storage and microgravity-related limitations may reduce germination.     

High performance capillary electrophoresis in microgravity environment.

In the last two decades, capillary electrophoresis has developed to a very quick and accurate analysis technique. With micellar techniques even uncharged particles can be analysed. The field of capillary electrophoresis is broad and shows an overlap with HPLC. The equipment for capillary electrophoresis is very compact, low in weight, energy consumption and waste production.     

Microcomputer-based monitoring of cardiovascular functions in simulated microgravity.

A microcomputer-based system for non-invasive monitoring of cardiovascular system in simulated microgravity is described. The system evaluates automatically, accurately and interactively heart beat intervals, beat-to-beat non-invasive finger arterial blood pressure (systolic, diastolic, mean and pulse pressure) using a Finapres device and beat-to-beat changes of thoracic blood volume using impedance changes. In addition, beat-to-beat evaluation of cardiac mechanical function including left ventricular ejection time, diastolic time, systolic time intervals, left ventricular ejection fraction estimate and several other contractility parameters, left ventricular volume, stroke volume and cardiac output estimates are performed with high degree of automaticity.     

A root moisture sensor for plants in microgravity.

Development of components for bioregenerative life-support systems is a vital step toward long-term space exploration. The culturing of plants in a microgravity environment may be optimized by the use of appropriate sensors and controllers. This paper describes a sensor developed for determining the amount of fluid (nutrient solution) available on the surface of a porous ceramic nutrient delivery substrate to the roots of conventional crop plants. The sensor is based on the change in thermal capacitance and thermal conductance near the surface as the moisture content changes. The sensor could be employed as a data acquisition and control sensor to support the automated monitoring of plants grown in a microgravity environment.     

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.     

The biological clock of Neurospora in a microgravity environment.

The circadian rhythm of conidiation in Neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). An experiment was conducted on space shuttle flight STS-9 in order to test this hypothesis; during the first 7-8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscillation, an increase in the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of flight, the tubes were exposed to light while their growth fronts were marked. Some aspect of the marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. These results from the last 86 hours of flight demonstrated that the rhythm can persist in space. Since the aberrant rhythmicity occurred prior to the marking procedure, but not after, it was hypothesized that the damping on STS-9 may have resulted from the hypergravity pulse of launch. To test this hypothesis, we conducted investigations into the effects of altered gravitational forces on conidiation. Exposure to hypergravity (via centrifugation), simulated microgravity (via the use of a clinostat) and altered orientations (via alterations in the vector of a 1 g force) were used to examine the effects of gravity upon the circadian rhythm of conidiation.     

Plant reproduction systems in microgravity: experimental data and hypotheses.

Elucidation of the possibilities for higher plants to realize complete ontogenesis, from seed to seed, and to propagate by seeds in microgravity, is a fundamental task of space biology connected with the working of the CELSS program. At present, there are results of only 6 spaceflight experiments with Arabidopsis thaliana, an ephemeral plant which will flower and fruit in orbit. Morphogenesis of generative organs occurs normally in microgravity, but unlike the ground control, buds and flowers mainly contain sterile elements of the androecium and gynoecium which degenerate at different stages of development in microgravity. Cytological peculiarities of male and female sterility in microgravity are similar to those occurring naturally during sexual differentiation. Many of the seed formed in microgravity do not contain embryos. Hypotheses to explain abnormal reproductive development in microgravity are: 1) nutritional deficiency, 2) insufficient light, 3) intensification of the influence of the above-mentioned factors by microgravity, 4) disturbances of a hormonal nature, and 5) the absence of pollination and fertilization. Possible ways for testing these hypotheses and obtaining viable seeds in microgravity are discussed.     

Lipid peroxidation of plants under microgravity and its simulation.

In series of space experiments aboard the biosatellites "Cosmos 1887", "Bion 9", the orbital stations "Salut", "Mir" and under clinostating, changes of lipid peroxidation (LPO) and antioxidation activity (AOA) of Chlorella, Haplopappus tissue culture, wheat and pea roots were determined. The changes had a complex fluctuation character; three steps of response were established: LPO decreasing accompanied by AOA increase; stabilization LPO <==> AOA balance; secondary LPO activation. Most early and highly amplitude decreasing of LPO were fixed in mitochondria. The rate of response have been increased on multicellular level of plants organization.     

Rhythmic biological systems under microgravity conditions.

Rhythmic phenomena in biology cover a wide frequency spectrum. In Space, the rhythms will encounter microgravity conditions which can, therefore, be a valuable tool for their understanding. A review and discussion of important effects of gravity/absence of gravity on biological systems will be given. Convection will be emphasized as a mechanism which is drastically reduced in Space. Microgravity might also affect the coupling between individual oscillators in a multi-oscillatory system. The environmental interference with rhythms will be discussed with a simple feedback as a starting point. Model simulations will be presented and clinostat and microgravity-conditions will be discussed in a specific case, viz. the gravitropical system of plants which can show sustained oscillations.