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.     

Possible use of a 3-D clinostat to analyze plant growth processes under microgravity conditions.

A three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles was constructed, and various growth processes of higher plants grown on this clinostat were compared with ground controls, with plants grown on the conventional horizontal clinostat, and with those under real microgravity in space. On the 3-D clinostat, cress roots developed a normal root cap and the statocytes showed the typical polar organization except a random distribution of statoliths. The structural features of clinostatted statocytes were fundamentally similar to those observed under real microgravity. The graviresponse of cress roots grown on the 3-D clinostat was the same as the control roots. On the 3-D clinostat, shoots and roots exhibited a spontaneous curvature as well as an altered growth direction. Such an automorphogenesis was sometimes exaggerated when plants were subjected to the horizontal rotation, whereas the curvature was suppressed on the vertical rotation. These discrepancies in curvature between the 3-D clinostat and the conventional ones appear to be brought about by the centrifugal force produced. Thus, the 3-D clinostat was proven as a useful device to simulate microgravity.     

Fluid physics experiments under microgravity conditions

The proposals for the detailed investigations of the Marangoni Bénard instability problem are described. It will be performed during the Spacelab D2 mission and prepared during Texus sounding rocket flight.     

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.     

Analysis of aggregation mechanism of erythrocytes under normal- and microgravity conditions.

Aggregation mechanism of erythrocytes under normal and microgravity conditions is analyzed from their recorded images. The video data is analyzed by PC/AT based image processing system. The results show that the shape of individual erythrocytes and their formed aggregates changes significantly which may affect the formation process of aggregates under microgravity conditions.     

Changes in the numbers of osteoclasts in newts under conditions of microgravity.

Intensity of osteoclastic resorption and calcium content were investigated in intact limb bones of the newts flown on board of a biosatellite Cosmos-2229 after amputation of their forelimbs and tail. Using X-ray microanalysis it was shown an increase in calcium content in the bones on 20th day after operation. Histological study revealed an activation of osteoclastic resorption on endosteal surface of long bones. The newts exposed after surgery on a biosatellite had the same level of bone mineralisation as operated ground control ones, but the increase in number of polynuclear osteoclasts was lower.     

Focal contacts organization in osteoblastic cells under microgravity and cyclic deformation conditions.

We compared quantitatively vinculin-related adhesion parameters in osteoblastic cells submitted to opposite mechanical stresses, i.e., low deformation and frequency strain regimens (stretch condition) and microgravity exposure (relaxed condition). Cyclic deformation induced a biphasic response comprising new focal contacts formation followed by their clustering in ROS cells. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. We previously demonstrated that 1% cyclic deformations at 0.05 Hz during a daily 10 min episode over 7 days stimulated ROS 17/2.8 growth as compared to static culture whereas relaxed ROS proliferated similarly to static culture (BC). To evaluate whether the proliferation (stretch) or the survival (relaxed) status of ROS cells influences focal contact organization, we inhibited ERKs proliferative-dependent pathway. Inhibition of proliferation by PD98059 was overcome although not fully restored by stretch. Furthermore stretch-induced clustering of vinculin-positive contacts still occurs in the presence of ERKs inhibitor, whereas the increase in focal contact number is abolished. In conclusion, we showed that focal contacts are mechanoeffectors and that hyper-mechanical stimulation could up regulate focal contacts size as compared to hypo-mechanical that down regulate clusterization.     

Development of tissue culture techniques and hardware to study mineralization under microgravity conditions.

To study the effects of weightlessness on mouse fetal long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with NA-beta-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type I experiment containers. The containers can be flushed with an air/5% CO2 gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of microgravity. Using 16 day old metatarsals the in vitro calcification process under microgravity conditions can be studied for a 4 day period.     

Growth of Prunus tree stems under simulated microgravity conditions

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees.     

Capillary brazing under microgravity (TEXUS-II) and 1g conditions

Experiments of vacuum brazing under both microgravity and 1-g conditions show the effect of hydrostatic pressure on ga-filling. The absence of buoyancy forces under microgravity affects the microstructure of the solidified braze in the joint.     

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.     

Peculiarities of genital organ formation in Arabidopsis thaliana (L) Heynh. under spaceflight conditions.

An experiment was carried out ahoard the Salyut 6 research orbital station on Arabidopsis thaliana cultivations. The seeds were sprouted in the Svetoblok 1 device which provides for plant growth in the agar medium under sterile conditions and at 4000 lux illumination. The experimental plants, as well as the controls, reached approximately the same developmental stages: both flowered and began to bear fruit. A microscopic examination of the generative organs in the control and experimental plants shows that in normally formed (by appearance) flower buds and flowers of the experimental plants, as distinct from the controls, there were no fertile elements of the adroecium and gynoecium. Degeneration of the latter occurred at different stages of generative organ development. Possible reasons for this phenomenon in plants grown under weightless conditions are considered.     

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.     

Mass transport in the near vicinity of solidification fronts under conditions of microgravity

Cellular solidification has been known and discussed since a long time. The appearance of cellular and dendritic microstructure closely resembles Benard cells known from fluid physics. Similar generation mechanismus may possibly be assumed. Both g-dependent and g-independent convective phenomena may probably be linked to the occurrence of instabilities at solidification fronts. It is to be expected that normal freezing of model alloys (advantageously such with no volume change at the freezing point) in a defined temperature gradient (gradient furnace) and quenching them may help to quantity g-influence on solidification.     

Changes of cell-vascular complex in zones of adaptive remodeling of the bone tissue under microgravity conditions.

We examined the peculiarities of the structure of the blood-vascular bed and perivascular cells in zones of osteogenesis in the epiphyses and metaphises of femoral bones of rats, flown aboard the US laboratory SLS-2 for two weeks by electron microscopy and histochemistry. In zones of bone remodeling, there was a tendency for a reduction of sinusoid capillary specific volume. Endotheliocytes preserve the typical structure. In the population of perivascular cells, we discovered differentiating osteogenic cells that contained alkaline phosphomonoesterase as well as cells that don't contain this enzyme and differentiate into fibroblasts. The fibroblasts genesis in zones of adaptive remodeling of spongy bones leads to a further development of fibrous tissue that is not subject to mineralization.     

Study of the Marangoni convection around a surface tension minimum under microgravity conditions

At equilibrium, aqueous fatty alcohol solutions presents a surface tension minimum versus temperature. The influence of such an extremum on the Marangoni convection is studied. Two experiments have been performed under microgravity conditions (Texus 8 (1983) and Texus 9 (1984) flights). The velocity fields are determined by following the paths of tracer particles and furthermore, in the Texus 9 experiment, differential interferograms have been recorded.     

Growth and development in higher plants under simulated microgravity conditions on a 3-dimensional clinostat.

Growth and development of etiolated pea (Pisum sativum L. cv. Alaska) and maize (Zea mays L. cv. Golden Cross Bantam) seedlings grown under simulated microgravity conditions were intensively studied using a 3-dimensional clinostat as a simulator of weightlessness. Epicotyls of etiolated pea seedlings grown on the clinostat were the most oriented toward the direction far from cotyledons. Mesocotyls of etiolated maize seedlings grew at random and coleoptiles curved slightly during clinostat rotation. Clinostat rotation promoted the emergence of the 3rd internodes in etiolated pea seedlings, while it significantly inhibited the growth of the 1st internodes. In maize seedlings, the growth of coleoptiles was little affected by clinostat rotation, but that of mesocotyls was suppressed, and therefore, the emergence of the leaf out of coleoptile was promoted. Clinostat rotation reduced the osmotic concentration in the 1st internodes of pea seedlings, although it has little effect on the 2nd and the 3rd internodes. Clinostat rotation also reduced the osmotic concentrations in both coleoptiles and mesocotyls of maize seedlings. Cell-wall extensibilities of the 1st and the 3rd internodes of pea seedlings grown on the clinostat were significantly lower and higher as compared with those on 1 g conditions, respectively. Cell-wall extensibility of mesocotyls in seedlings grown on the clinostat also decreased. Changes in cell wall properties seem to be well correlated to the growth of each organ in pea and maize seedlings. These results suggest that the growth and development of plants is controlled under gravity on earth, and that the growth responses of higher plants to microgravity conditions are regulated by both cell-wall mechanical properties and osmotic properties of stem cells.     

First results of the growing of PbTe single crystals under microgravity conditions

Two sublimation experiments with PbTe were performed at 850°C and 750°C under microgravity conditions during the “Salyut-6”-“Soyuz-31” mission in 1978. The sublimation took place on the (100)-face of a PbTe crystal. The condensate grown at 850°C shows in the upper part the formation of a pyramidal habit. The bulk consists of parallel growth colums with [100]-boundary faces. The charge carrier concentration of the electrons (N=1,5·10¹⁹ cm^?3) is higher than the homogeniety limit for 800°C should permit. This is in contrast to the result obtained on the PbTe crystal of the parallel experiment on the earth. At 750°C a laminated condensate is grown with a low growth rate. The microphotograph of the substrate shows the beginning whisker formation according to the VLS-mechanism.     

Development of a plant growth unit for growing plants over a long-term life cycle under microgravity conditions.

To study the effect of the space environment on plant growth including the reproductive growth and genetic aberration for a long-term plant life cycle, we have initiated development of a new type of facility for growing plants under microgravity conditions. The facility is constructed with subsystems for controlling environmental elements. In this paper, the concept of the facility design is outlined. Subsystems controlling air temperature, humidity, CO2 concentration, light and air circulation around plants and delivering recycled water and nutrients to roots are the major concerns. Plant experiments for developing the facility and future plant experiments with the completed facility are also overviewed. We intend to install this facility in the Japan Experiment Facility (JEM) boarded on the International Space Station.     

Repair of radiation induced genetic damage under microgravity.

The influence of microgravity on the repair of radiation induced genetic damage in a temperature-conditional repair mutant of the yeast Saccharomyces cerevisiae (rad 54-3) was investigated onboard the IML-1 mission (January 22nd-30th 1992, STS-42). Cells were irradiated before the flight, incubated under microgravity at the permissive (22 degrees C) and restrictive (36 degrees C) temperature and afterwards tested for survival. The results suggest that repair may be reduced under microgravity.