POSTER SESSION

Magnetic resonance microscopy for the quantitative analysis of osteoporotic bone

R.Toffanin^{1, 2}, A.Accardo³, M.Cova⁴ and F. Vittur^1
1 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy ² PROTOS Research Institute, P.O. Box 972, I-34100 Trieste, Italy ³ Department of Electro-Technics, Electronics and Informatics, University of Trieste, Via Valerio 10, I-34127 Trieste, Italy ⁴ Institute of Radiology, University of Trieste, Cattinara Hospital, Strada di Fiume 447, I-34149 Trieste, Italy

The skeleton comprises two different forms of bone: cortical bone and trabecular bone. Cortical bone serves primarily mechanical and protective functions and also provides skeletal strength. Trabecular bone, in addition to providing mechanical and skeletal strength, plays an important metabolic function. Bone disorders such as osteoporosis lead to alterations in the trabecular bone. These alterations are not only characterized by a reduction of bone mass but also by structural changes in the architecture of bone. An accelerated bone loss together with a bone structure impairment, especially in the weight-bearing bones, are also observed on astronauts and animals exposed for prolonged periods to microgravity. This problem, which involves a high risk of bone fracture, is of great relevance to the various space agencies (NASA, ESA, etc.) in view of the forthcoming utilization of the International Space Station. As a consequence, there is great need to develop accurate methods for monitoring the loss of bone mass on astronauts during prolonged spaceflight and for evaluating the trabecular bone architecture after returning to earth. These methods not only would help improve health and safety of astronauts aboard the Space Station but also would permit a better understanding of the structural changes that bones undergo in the presence of microgravity and their readaptation upon returning to planetary gravity levels. The in vivo observation of the bone architecture is today clinically not feasible but the use of magnetic resonance imaging (MRI) techniques in the area of osteoporosis appears very promising. Among these techniques, magnetic resonance microscopy (MRM) is certainly one of the most powerful methodologies for the ex vivo study of trabecular bone being able to provide three-dimensional information of the bone architecture. However, the trabecular width derived from conventional MR images is generally overestimated as the susceptibility effect at the bone-marrow interface causes signal dephasing. Such an effect can be minimized if the echo-time (TE) or voxel size are reduced. The purpose of our research was the development of new MRM techniques that have a potential role in the characterization of trabecular bone architecture. In this study we describe the use of short-TE projection reconstruction MRM for the study of normal and osteoporotic bone explants. This method promises to be more accurate than conventional MRM in the quantitative analysis of trabecular bone. In vivo projection reconstruction MR imaging could be applied to evaluate bone architecture and bone quality evolution after space flight exposure.

Effectiveness of the Random Positioning Machine (RPM) to provide simulated microgravity for cultured fetal mouse long bones

J. P. Veldhuijzen, J. M.A. de Blieck-Hogervorst and J.W.A. van Loon
Dept. of Oral Cell Biology, ACTA - Vrije University, Amsterdam, The Netherlands

Having the possibility to perform experiments in the laboratory under simulated microgravity conditions would be a great advantage in microgravity research. Recently a Random Positioning Machine has been developed (Fokker Space, The Netherlands) which should offer simulated microgravity conditions. Previous experiments with cultured fetal mouse long bones under space flight conditions (IML-1, IML2 and BION-10) revealed that 4 days of real microgravity did not change overall growth but reduced the mineralization of the diaphysis. Effectiveness of the RPM to duplicate the results of our flight experiments was tested in 4-day cultures of 17-day old fetal mouse long bones in real flight hardware. The long bones were individually cultured in double-layered culture bags with 0.7 ml medium (alfa-MEM including 5% FCS and 2-3 mM Na-ß-glycerophosphate). Experiments were performed in Biorack type I/0 containers, each with 8 culture bags. The content of the container was flushed with air/5% CO₂ at the beginning of the experiment and, in a number of experiments, again after 2 days. Prior to the experiment and at the end, photomicrographs were taken of each individual long bone. These photographs were used for measurements to calculate the increase in overall length and length of the mineralized diaphysis.
In space flight experiments the isolated long bones are not attached inside the culture bag but in the RPM this resulted in movements of the long bones that should be avoided in true random positioning. Experiments with these "mobile" long bones did not show any difference in growth and mineralization between the RPM and control cultures. We were able to prevent these movements by immobilizing the long bones in a small piece of agarose gel. Overall growth was never changed under RPM-conditions. In most of the experiments also no differences in the mean values for mineralization on the RPM and under control conditions were found. However in the RPM-groups in many cases the standard deviation was higher then in the controls. In some experiments comparable results were found as under real microgravity conditions (decreased mineralization), whereas in another experiment the mineralization seemed to have increased on the RPM.
The inability to reproduce the results of our flight experiment on the RPM leads to the conclusion that the RPM did not provide simulated microgravity to our fetal mouse long bones. However during the introduction of the agarose-immobilized long bones into the culture bags it was in most cases impossible to avoid also the inclusion of air bubbles. These air bubbles do not affect culture conditions under real microgravity or in the controls of the RPM-experiments. In the culture bags on the RPM however, these bubbles are very motile during RPM-operation and may have mixed the medium, resulting in different culture conditions, which may have obscured possible differences between the RPM-groups and the controls. Further experiments are needed to clarify this issue.
This project was supported by the Space Research Organization of the Netherlands (SRON grant MG-045)

A PROJECT PROPOSAL IN SPACE BIOLOGY: EXPOSURE TO MICROGRAVITY AS A POWERFUL TOOL TO STUDY THE LIFETIME-LONG SENESCENCE PROCESS IN AN ACCELERATED TIME-SCALE

P. Degan
National Cancer Research Institute-Genova - 16132 GENOVA – ITALY

Microgravity mimes on a many-fold compressed time-scale the effects of ageing and senescence. Since these changes normally happen during extended times, an extraordinary opportunity in space research is the possibility to produce suitable experimental protocols that allow to mime generational-long processes in a model system otherwise unavailable for the study of ageing.
During senescence a number of biological processes are lengthened and stretched during an expanded time-scale. Metabolic rate, blood circulation, proliferative ability and tissue regeneration are reduced. These same alterations have been found in cells and organisms subjected to microgravitational conditions. Maintenance of microgravitational conditions results in profound changes in the utilization of different metabolites, in the maintenance of an energetic balance and in the increase of glucose utilization, maybe associated with a lower ability to produce ATP from mitochondria. As a whole these changes reflect profound alterations in the expression and control of a wide array of genes as it happens in pathological degenerative processes as well as in the ageing process.
A very simple and straightforward approach is proposed to follow the fluctuation of a wide series of genes involved in the modulation of growth conditions relevant to specific sets of stress. Gene expression array systems-like technologies will be employed to evaluate if specific gene expression profiles are modified following these treatments.
This experimental framework can provide the knowledge for the development of pharmacological and therapeutic tools in the treatment of degenerative processes, a particularly demanding issue to our society in terms of economic and health costs.
This report intends to be a provocative introduction to a project proposal aimed to study senescence obtained after exposure to microgravity of normal cells or cells from patients affected by congenital diseases showing a premature senescence phenotype or normal cells transfected with genes that are typically expressed in these diseases.

The effect of microgravity on cell functions related to chromosome segregation and oxidative stress

P. Degan, C.F. Cesarone*, L. Ottaggio, A. Zunino, S. Viaggi°, P. Pippia*, G. Galleri*, M.A. Meloni*, S. Bonatti°, A. Abbondando°
National Cancer Research Institute-Genova - I-16132 GENOVA – ITALY °CNR Institute of Mutagenesis and Differentiation – I- 56010 PISA – ITALY °Department of Oncology Biology and Genetics - University of Genoa – I-16132 GENOVA ITALY. *Department of Physiology, Biochemistry and Cell Sciences - University of Sassari I-07100 SASSARI - ITALY

Spaceflight experiments have shown that a number of cell functions including cell growth, cell proliferation, secretory capacity, differentiation and development are altered during the exposure to microgravity (0g). Two distinctive cell functions
1) the segregation of chromosomes at mitosis and 2) the handling of DNA damage and repair including oxidative stress, have received so far relatively little attention. We thought that these functions should be studied in simple and well characterized cell systems, before moving to more complex in-vivo systems. This is the framework of our project supported during the last year by the Italian Space Agency (ASI).
Chromosome segregation and oxidative metabolism are relevant functions from the standpoint of human health. It is well established that an increased rate of errors in chromosome segregation is associated to an increased risk of cancer and genetic diseases. Alteration of the oxidative metabolism has been related to degenerative diseases and ageing and oxidative stress is an important modulator of gene activity. Interestingly, microgravity has been found to affect a number of signal transduction pathways within the cell that are involved in cell cycle control, cell proliferation and apoptosis.
Alterations to the cellular metabolism induced by exposure to 0g are evidentiated in the modification of the PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to 0g in a Random Positioning Machine (RPM) in complete medium for different periods.
The cellular ATP content are strongly affected by exposure to µg for 24, 36 or 48 h. It is not yet clear whether this may be attributed to a decreased production or to an enhanced utilization of pre-existing intracellular ATP in a condition in which cellular metabolism (and specifically the mitochondrial oxidative phosphorilation) is somehow impaired. In these condition PARP activity remains low, an indication that no gross metabolic unbalance neither DNA damage occurs under these conditions. However treatment of the cells with a strong oxidant (KBrO3) does induce an immediate induction of PARP. While either control or 0g exposed cells show the same susceptibility to this damaging agent, either the relevant increase in 8-OHdG immediately after the removal of the agent and the slow
repair process of this adduct suggests that important alterations in cellular metabolism are associated with the exposure to µg.

MAIN RESULTS OF THE FERTILE EXPERIMENT PERFORMED ONBOARD THE MIR SPACE STATION USING PLEURODELES WALTL, AN URODELE AMPHIBIAN

H. Membre¹, L. Gualandris-Parisot², D. Durand¹, A. Bautz¹ and Ch. Dournon^1
1EA 2401: Genetic, Signalization, Differentiation; Laboratory of Experimental biology and Immunology; Faculty of Sciences, Henri Poincaré University, Nancy, France. 2Centre de Biologie du Développement, UMR 5547 CNRS-Université Paul Sabatier, Toulouse, France.

The FERTILE experiment was twice performed onboard the MIR space station. The first experiment occurred during the 1996 French Cassiopée mission from August 17 to September 2, and the second one during the 1998 French Pégase mission from January 29 to February 19. The aim of the study was to analyze in the urodele amphibian Pleurodeles waltl, microgravity effects on the fertilization and embryonic development, and then to study the further development after return on ground. For this experiment, an instrument called Fertile was developed by the CNES and used to rear numerous eggs, embryos and adults on trays in µG conditions, and several eggs and embryos in a 1G-rotating centrifuge. The development was studied using combination of video recording observations, morphological and SEM analyses of whole embryos, and histological and immuno-histological sections of fixed embryos. Moreover, some embryos were kept alive after landing. Genetic and cytomorphologic analyses proved that an actual fertilization occurred in µG. Nevertheless, some characteristic processes of the fertilization were altered. During the early development, abnormalities corresponding to cortical cytoplasm movements, cell adhesion decrease and loss of cells were observed. In several embryos, the cleavage was irregular and the neural tube closed with difficulties. These temporary abnormalities were regulated during the following embryonic stages, and young larvae with normal morphology and behavior hatched in microgravity as control animals on earth. Recovered on ground at post-hatching stage, young larvae were reared at room temperature, underwent metamorphosis and became mature without obvious abnormalities. The rates of development and morphology were analogous for these animals and for ground controls reared during a similar annual period. Analysis of offspring was performed. Born-in-space males were firstly mated with ground-control females, and then with born-in-space females. The mating gave progenies that normally developed. Depending on the techniques used and on the limits of analyses, the study clearly demonstrated that these amphibian embryos born in space are able to live and to reproduce after return on earth.
- Gualandris-Parisot L, Bautz A, Chaput D, Husson D, Durand D, Dournon C (1998) Mises au point technologiques en vue d’étudier le développement du Pleurodèle (Amphibien Urodèle) à bord de la station spatiale MIR. Récents Progrès en Génie des Procédés 62: 37-48.
Aimar C, Bautz A, Durand D, Membre H, Chardard D, Gualandris-Parisot L, Husson D, Dournon C (2000) Microgravity and hypergravity effects on fertilization of the salamander. Biology of Reprodroduction 63: 551-558.
- Dournon C, Durand D, Tankosic C, Membre H, Gualandris-Parisot L, Bautz A (2001) Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl. Development Growth and Differentiation 43: 315-326.
- Gualandris-Parisot L, Husson D, Bautz A, Durand D, Kan P, Aimar C, Membre H, Duprat AM, Dournon C (2001) Microgravity effects on the embryonic development up to hatching stage of in flight fertilized salamander eggs. Development, Genes and Evolution (accepted). This work was granted by the CNES

REFERENCE VALUES OF ENZYMATIC ACTIVITIES IN PLEURODELES WALTL FOR FUTURE SPACE MISSIONS

T. Alekhova¹, A. Sofin¹, T. Kobelkova¹, T. Novozhilova¹, R. Marco² and Ch. Dournon^3
1A.N. Bach Biochemistry Institute of the Russian Academy of Sciences, Moscow, Russia. ² Department of Biochemistry of the Autonomous University of Madrid, Instituto "Alberto Sols", Faculty of Medicine, Madrid, Spain. ³ EA 2401: Genetic, Signalization, Differentiation; Faculty of Sciences, Henri Poincaré University, Nancy, France.

To prepare a space experiment using Pleurodeles waltl (urodele amphibian), the objective of this study was to establish in adults reared on ground at standard laboratory conditions the reference values of the activity of several blood enzymes involved in cell destruction, stress response or used as indicators of cell metabolism. The final goal of this experiment is to obtain reproduction and long-term development onboard a space station. For this preliminary evaluation, we selected adults during the reproductive period. The reference values of the activity of several blood enzymes will be used later to estimate and to compare the physiological and biochemical status of this amphibian when reared under different environmental situations, and particularly under extreme temperature or microgravity conditions. Through the use of plasma and erythrocytes, several samples from the same live animal will be obtained. The selected enzymes were glutamate dehydrogenase, aspartate aminotransferase, alanine amino-transferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase.
For most enzymes, we established that the activity level was different in females and males. In fresh samples of blood plasma, the enzyme activities in females were higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males, and for glutamate dehydrogenase activity, which was higher in males than in females. In female erythrocytes, the average level of activity for all the studied enzymes was higher than in male erythrocytes.
Freshly isolated, frozen or thawed samples of plasma and erythrocytes were also studied to define the best conditions of storage taken off in microgravity during a space mission, and on ground before and after this mission. For most enzymes, the activities in freshly isolated plasma and erythrocyte preparations decreased after a storage at -18oC or at +4oC. Freshly isolated samples gave the best results. However, the protocol of sampling for a space mission needs storage until the analysis on-ground takes place a few months later. Several improvements in storage will be tested in the future. Among them, we propose the incorporation of proteolytic inhibitors in the preparation buffers, addition of cryoprotectant agents to prevent freezing, use of faster procedures of cooling and/or warming, as well as lower temperatures of storage.
This work was supported by the international grant INTAS-CNES No 97-1060

EFFECTS OF MICROGRAVITY AND HYPERGRAVITY ON EARLY DEVELOPMENTAL STAGES OF XENOPUS LAEVIS: MORPHOLOGICAL AND FUNCTIONAL ASPECTS

A.M. Rizzo, F. Rossi, A., Guerra, P. Pippia*, M.A. Meloni* and B. Berra
Institute of General Physiology and Biochemistry, University of Milan,
Via D. Trentacoste 2, 20134 Milan ITALY. Department of Physiological Biochemical and Cellular Sciences, University of Sassari, Via Muroni 25, 07100 Sassari.

The aim of this work is to evaluate the development of Xenopus laevis in modified gravity conditions studying: 1- structural and functional components of the cell membrane such as phospholipids, cholesterol, neutral and acidic glycolipids; 2- the effect of the peroxidative stress after experimental radiation exposure; 3- the modification of the motility due to gravity conditions. It is well known that glycolipids (in particular gangliosides) are involved in neuronal development and functioning. The different phospholipid classes and their reciprocal relative concentration together with the amount of glycolipids and cholesterol play an important role in the physico-chemical properties of the cell membrane. We are also interested to evaluate, during embryonic development, the enzyme activities involved in glycolipid metabolism including the regulation of their expression; our aim is to investigate the possible involvement of modified gravity conditions on these parameters.
The literature on Xenopus laevis lipid content and distribution was scarce; for this reason in these last years we focused our research on early stage of Xenopus laevis development studying the content of the major lipid classes as neutral lipids, phospholipids and glycolipids and their metabolism.
Xenopus laevis was one of the first organism in which the processes of fertilization and embryogenesis were studied in microgravity conditions. Several papers are present in the literature demonstrating the influence of modified gravitational forces on the basic behavioural features of lower aquatic vertebrates. These observations should be considered by neurobiologists as a provocative starting point for the elucidation and interpretation, on a biological basis, of the above mentioned changes. No changes were found so far in content, composition and distribution of ions under near weightlessness conditions; on the contrary significant alterations in the activity of various enzymes related to brain energy metabolism and neuronal membrane constituents were observed. Some authors clearly demonstrated subtle changes in brain metabolism in order to respond to changes in the gravity environment. In particular the AA observed that the exposure of fish larvae to hyperpergravity conditions caused variations in the composition of glycoconjugates together with a slight increase of the activity of sialidase, enzyme involved in the catabolism of glycoconjugates itself. It is worth noting that these compounds have been investigated in our laboratory for many years. These studies demonstrate that some animal models are very sensitive to gravitational modifications and react to this "stress" by modifying their metabolism, which supports our hypothesis.
On this basis we utilized Xenopus laevis embryo, to evaluate the effects of the simulation of micro and/or hypergravity, performed in Sassari (Sardegna, Italy) utilizing an hyperfuge and a 3d Clinostat (Random Positioning Machine, RPM)
The experimental protocol, considering that X.l. embryo development is characterized by a hatching period, of 72 hrs post fertilization (p.f.) was the following:
Group A: 8 hrs after in-vitro fertilization the embryos were placed on the RPM till day 6 of development.
Group B: 8 hrs after in-vitro fertilization the embryos were placed on the RPM till day 3 of development.
Group C: an other set of embryos was subjected to microgravity after hatching (from day 3 of development) until day 6.
In parallel Xenopus embryos were subjected to hypergravity (1.5-2 g) using the centrifuge, following the time protocol defined for the clinostat.
At the end of the experiments morphological studies and lipid analysis were performed, as previously described (2-4). Some biochemical changes have been observed in the embryos kept in the clinostat and hyperfuge compared with the controls, demonstrating that, even if no gross malformations are present, embryo development is influenced by the gravity condition.
In conclusion we would like to stress the usefulness of this animal model for the kind of studies that allows to investigate the effects of gravitational fields on a live vertebrate animal, and to better comprehend the importance of these forces for embryonic development. The aim of our future research is to also study the influence of peroxidative stress in modified gravity on complex lipid metabolism.
REFERENCES
1-K. Slenzka et al. Proceedings 5th Europ. Symp. On "Life Sciences Research in Space" Arcachon France 26 Sept 1993.
2-Gornati R., Rizzo A.M., Tong X.W., Berra B. and Bernardini G. (1995). Glycolipid pattern during Xenopus embryo development. Cell Biol. Int. 19, 183-189.
3-Rizzo A.M., Gornati R., Galli C., Bernardini G. and Berra B. (1994). Cholesterol, triacylglicerols and phospholipids during Xenopus embryo development. Cell Biol. Int. 18: 1085-1090.
4-Rizzo A.M., Gornati R., Rossi F., Bernardini G. and Berra B. (1995). Retinoic acid induces changes in Xenopus embryo glycolipid pattern. Cell Biol. Int. 19, 895-901.

GROWTH OF FISH INNER EAR OTOLITHS IS GUIDED BY THE GRAVIVECTOR AND REGULATED BY THE CNS

R. H. Anken, M. Beier, E. Edelmann and H. Rahmann
Zoological Institute, University of Stuttgart-Hohenheim, Garbenstr. 30, D-70593 Stuttgart, Germany

BACKGROUND: Recent investigations were conducted in order to test the hypothesis that the "physical" capacity (i.e., weight, size) of an inner ear stone (otoconial mass called statolith, which consists mainly of calcium carbonate) on the sensory epithelium could be a regulating factor in controlling the growth of this mass via a negative-feedback loop between the brain and the inner ear. These analyses, having been performed on mammalian and avian species, however, yielded contradictory results.
AIM OF THE STUDY: In contrast to other vertebrates, fish possess compact otoliths, the weight or/and size of which can be easily assessed quantitatively. We were thus prompted to investigate otolith growth under hypergravity (3g, centrifuge) conditions. Another experiment involved the unilateral transection of the vestibular nerve in order to clarify whether the efferent vestibular system connecting the brain with the inner ear plays a role in otolith growth.
MATERIALS AND METHODS: In the first experiment, otoliths of freshly hatched cichlid fish Oreochromis mossambicus (Perciformes) were labelled with the calcium-tracer alizarin-complexone (AC) at 1g earth gravity before and after a 3, 7, 14 or 21 days stay of the animals at hypergravity conditions (hg; 3g, centrifuge). After the experiment, the otoliths´ area between the two AC-labellings was measured with regard to size and asymmetry (size difference between the left and the right stones). In the course of the second experiment, the vestibular nerve was unilaterally transected in neonate swordtails (Xiphophorus helleri, Atherinoformes), which were subsequently incubated three times in AC (immediately after nerve transection and again after 7 and 14 days).
RESULTS AND DISCUSSION: Both utricular and saccular otoliths (lapilli and sagittae, respectively) continued growing in a linear way at hg, but already at day 7 growth was highly significantly slowed down as compared to parallely raised 1g-control specimens. In the case of bilateral asymmetry between the corresponding otoliths its formation in hg-animals became reduced as compared to the 1g controls. The reduction of asymmetry was much more pronounced in the sagittae than in the lapilli. The latter result supports an earlier hypothesis, according to which especially a low sagittal asymmetry has a functional advantage. The densitometric quantification of AC deposited on the otoliths after vestibular nerve transection resulted in significantly lower AC-values in the otoliths of the transected side (either right or left), whereas the controls (either irritated unilaterally or nontransected) did not reveal any significant differences in otolithic AC-density on both sides of the body. After 14 days, AC was again incorporated into the otoliths, which indicates that the vestibular nerve had regenerated.
CONCLUSION: The results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector. Since the otolithic calcium incorporation ceased on the transected head sides, it is concluded that the regulation of otolith growth is based on the central nervous efferent vestibular system. It has now to be clarified whether these mechanisms are also responsible for statolith growth in higher vertebrates including humans.
ACKNOWLEDGEMENT: This work was financially supported by the German Aerospace Center (DLR) e.V. (FKZ: 50 WB 9997).

OTOLITH ASYMMETRY AND INNER EAR MORPHOLOGY OF MOTION SICK FISH: A PARABOLIC AIRCRAFT FLIGHT STUDY

R. Hilbig, R. H. Anken, G. Sonntag, N. Kretschmer, A. Bäuerle, N. Baumhauer, M. Knappenberger, and H. Rahmann
Zoological Institute, University of Stuttgart-Hohenheim, Garbenstr. 30, D-70593 Stuttgart, Germany

BACKGROUND: Humans taking part in parabolic aircraft flights (PAFs) may suffer from (space) motion sickness (SMS, a kinetosis). Following a theoretical concept, susceptibility to SMS may be based on asymmetric inner ear statoliths (i.e., differently weighed statoliths on the right and the left side of the head) with asymmetric inputs to the brain being disclosed at microgravity.
AIM OF THE STUDY: Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated whether kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry. Further investigations were focused on the morphology of the inner ear.
MATERIALS AND METHODS: Swordtails (Xiphophorus helleri, Atherinoformes) aged 15 - 21 days after birth and cichlids (Oreochromis mossambicus, Perciformes) aged 24 – 30 days post fertilization at 22°C were subjected to PAFs (A 300 ZERO-G/NOVESPACE). The hardware was designed to perform video controlled acceleration experiments, allowing the analysis of the behaviour of individual animals. A group of swordtails had been tested pre-flight concerning their performance of the dorsal light response (DLR). These animals were then grouped into individuals relying either more on their visual or on their vestibular system for spatial orientation. After the PAFs, otoliths were measured planimetrically and inner ear sensory epithelia were examined qualitatively and quantitatively (cell countings) on the light microscopical level.
RESULTS AND DISCUSSION: During microgravity, some 10% both of the cichlid larvae and the neonate swordtails exhibited a kinetotic behaviour, whereas the remaining animals swam normally or were disorientated (zig-zag-swimming). There was a significant difference in the behaviour of swordtails, who had shown (pre-flight) either a poor or an elaborate DLR-performance in terms that the individuals of the latter group exhibited a reduced kinetotic behaviour since they obviously preferred visual cues for postural control.
The analysis of the otoliths revealed a 99% correlation (p<0.01) of a particular behaviour (i.e., normal or kinetotic swimming) with the amount of (individual) otolith asymmetry, which was especially pronounced in kinetotically swimming individuals. This finding speaks in favour of the aforementioned asymmetry-hypothesis as the main source for kinetosis-susceptibility. On the histological level, however, it was found that the cell density of macular sensory epithelia (sensory and supporting cells/100µm²) was reduced in kinetotic animals as compared to normally swimming fish, which seemed to be due to a possibly pathological increase in cell size of the kinetotic specimens. Thus, an individual fish, who swam kinetotically, needed not necessarily to have asymmetric otoliths but could have had morphologically aberrative maculae and, vice versa, fish with highly asymmetric otoliths needed not always to be susceptible to kinetosis if they preferred rather visual than vestibular cues for orientation.
CONCLUSION: Asymmetric otoliths can cause kinetosis in fish during PAFs, but susceptibility to kinetosis may also be based on an aberrative inner ear morphology. The individual preference of visual or vestibular cues (DLR performance) seems further to be of crucial importance in this context, which is in concordance with findings on humans.
ACKNOWLEDGEMENT: This work was financially supported by the German Aerospace Center (DLR) e.V. (FKZ: 50 WB 9997).

A MOUSE MODEL FOR THE EVALUATION OF NEUROBEHAVIOURAL RESPONSES TO HYPERGRAVITY

D. Santucci, N. Francia, G. Corazzi, L. Aloe* and E. Alleva
Behavioural Pathophysiology Section, Lab. Fisiopatologia di Organo e di Sistema, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Roma (Italy) and * Istituto di Neurobiologia, Viale Carlo Marx 15, I-00137 Roma (Italy)

In mammals, alterations in vestibular functions due to modification of the gravitational environment cause motion sickness (MS). In non-emetic vertebrates, such as rodents, pica behaviour (i.e. consumption of non nutritive substances such as kaolin) has been considered an appropriate index of MS. We investigated behavioural responses to hypergravity in CD1 mice during late postnatal development and at adulthood. Post-weanling (postnatal day 28, PND 28), adolescent (PND 42), young-adult (PND 60) and adult male and female mice were exposed to 2g rotationally-generated hypergravity for a single 60 or 120 min session. Pica behaviour, measured through kaolin consumption, and ethological-type scoring of different activities were evaluated before, during and after exposure. Furthermore, the short- and/or long-lasting effects of exposure on emotional responses (plus-maze test), exploratory behaviour (hole-board test) and spatial learning performances (Morris Water Maze test) were investigated. Finally, in order to correlate behavioural changes with alterations in central levels of neurotrophins, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) levels were assessed. Pica behaviour increased during post-rotation while a reduction in spontaneous activity during rotation indicated a transient mild sickness associated with hypergravity. Long-lasting effects of exposure were observed, rotated mice showing altered emotional/anxiety behavioural profiles. Central NGF levels were markedly increased after rotation while no major changes were observed in BDNF expression. The mouse appears to be a suitable species for investigating motion sickness syndrome as well as more general adaptive responses to altered gravitational stimuli. Ground-based hypergravitational studies on this small sized mammalian species may therefore be useful in the light of the increasing numbers of space missions. Moreover, in view of the relevance of the mother-infant relationship to allow mammals development in space, we start to study the effects of 2g exposure on maternal behaviour as well as on pups development.

EFFECTS OF MICROGAVITY AND ETHYLENE ON CELL ULTRASTRUCTURE AND MOBILIZATION OF STORAGE METABOLITES FROM COTYLEDONS

O. M. Nedukha¹ and Ch. S. Brown^2
1Department of Cell Biology, Institute of Botany, 01004, Kiev, Ukraine, ²Dynamac Corp., and NSCORT, NC State University, USA

Six-days soybean seedlings (Glycine max L. [Merr.]) were planted from dry seeds onboard of the space shuttle Columbia (STS, December 1 – December 7, 1997) and in the ground control in NASA laboratories at Kennedy Space Center. The seedlings were cultivated in BRIC (Biological Research in Canisters) hardware in darkness. The packets with purafil (KMnO₄) were placed into half of the canisters for the removing of some part of ethylene that secretes from seedlings. Live seedlings were used for cytological study and biochemical determination of lipid content after landing. The middle parts of cotyledons were fixed for light, electron microscopical, and electron-cytochemical study. The pyroantimonate method was used for the study of ionized calcium localization. We had four variants of seedlings: ground control (-purafil), ground control (+purafil), microgravity (-purafil) and microgravity (+purafil). It is established that the level of ethylene was almost 1,5 times more in flight canisters in comparison with that in control canisters. Light microscopical study of the storage mesophyll cells structure in the cotyledons did not reveal essential differences in the investigated seedlings of all variants. Lipid content in seedling cotyledons of ground (+purafil) variant equaled 22,1±0,8; in seedling cotyledons of ground
(-purafil) variant - 22,2±0,9 mg/100mg dry weight. Lipid content equaled 19,4±0,7 and 18,8±0,4 mg/100mg in seedling cotyledons of flight (+purafil) and of flight
(-purafil) accordingly. Ultrastructure of mesophyll cells was typical for cells of storage tissue in cotyledons of soybean seedlings of two variants of ground control. Vacuoles contained fibrillar components. Such vacuoles contained storage protein (according to literature data). A lot of cytoplasmic electron dense lipid bodies occupied a large part of hyaloplasm. Lipid bodies had round forms in sections and were from 0,3 to 1,5 μm in diameter. A large part of lipid bodies had the signs of lysis. Amyloplasts and leukoplasts presented plastidom. Plastids contained large starch grains, prethylakoid body, small plastoglobules and light stroma. Endoplasmic reticulum was canalicular. Large mitochondria had weakly developed cristae. Ionized calcium was revealed in vacuoles, hyaloplasm, interspaces and all organelles. Differences of cell ultrastructures of tissue of the ground control (-purafil) seedlings and that of ground control (+purafil) seedlings were expressed by the clarifying of vacuoles and the presence of a developed Golgi apparatus and a rough endoplasmic reticulum, and the decrease of ionized calcium in interspaces. The ultrastructure of most parts of cotyledon mesophyll cells in flight seedlings is independent from the presence of purafil in the canisters compared to ground control cells. There was a reduction of size and quantity of cytoplasmic storage lipid bodies, and a decrease of ionized calcium in hyaloplasm, vacuoles and in interspace. The results show that microgravity had an influence on the utilization acceleration of both storage cytoplasmic lipids and possible storage vacuolar proteins from cotyledons during growth of soybean seedlings independently from the exogenous ethylene level in the canisters.

LOW GRAVITY AND ENZYMATIC ACTIVITIES: EFFECT OF SIMULATED MICROGRAVITY ON PARP-1 ACTIVITY IN HUMAN LYMPHOCYTES AND IN RAT HEPATOCYTES

C.F. Cesarone, P. Pippia, M.A. Meloni, G. Galleri, M.G. Camboni, L. Demori, L. Scarabelli, E. Fugassa and A. Cogoli
Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari, Università di Sassari, Via Muroni 25, I- 07100 Sassari ° Dip. Biologia Sperimentale, Ambientale e Applicata, Università di Genova *Space Biology Group, ETH, CH-8005 Zurich

Experiments conducted in space in the last two decades have shown that the mitogenic in vitro activation of human T lymphocytes is remarkably reduced by microgravity. Recently, using the Random Positioning Machine (RPM) to simulate microgravity, we have found direct gravitational effects on the gene expression of interleukin-2 and its receptor in T cells. Moreover, several experiments performed in space have demonstrated that low gravity can lead to significant changes on a number of physiological functions such as cell differentiation, signal transduction, gene expression and DNA metabolism. In these processes, a basic role is played by poly(ADP-ribosyl)ation, a post-transcriptional modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP). Member of PARPs family, PARP-1 is the abundant chromatin-bound enzyme, which transfers the ADP-ribose units from NAD+ to acceptor proteins, providing also for automodification.
Human peripheral blood lymphocytes (PBL), isolated from healthy donors and separated by density gradient centrifugation on Ficoll-Histopaque, and rat hepatocytes, isolated by a standardized collagenase perfusion technique, were exposed for 24 hours to simulated microgravity produced by the RPM. The PARP-1 activity was determined in isolated, permeabilized human PBL and in cultured hepatocytes monolayers by evaluating the incorporation rate of labelled ADP-ribose, from [³²P]NAD (5 µCi/nmol) into acid-insoluble material. The total level of the catalytic protein was evaluated, in cell extracts, by the activity gel technique, which allows the autoradiographyc detection of PARP-1 activity after SDS-PAGE and in situ renaturation of the proteins. DNA content was determined fluorometrically by micromethod using 33258 Hoechst dye. In addition, to evaluate the possible effects on hormone-mediated gene expression in rat hepatocytes, a separate set of cell monolayers were treated with 10^-6 M triiodo-L-thyronine (T₃) during µG exposure. When measured, the relaxating activity of DNA topoisomerase I (Topo I), which is among the nuclear targets under PARP-1 control, was evaluated by reacting cell extracts with supercoiled pBR322 phage DNA. Topoisomers were then separated by 0.8% agarose gel electrophoresis, stained with Et-Br and analyzed by densitometry.
Preliminary results show that in Concanavalin A-treated and untreated PBL exposed for 24 h to microgravity PARP activity is partially inhibited (35-40%), when compared to control unexposed cells. This inhibition is probably among the earliest outcomes of a more complex series of events culminating in the adaptive response observed after long-lasting exposure to hypogravity. Instead, rat hepatocytes exposed to low-g conditions revealed a higher activity level of PARP, when compared to control unexposed cells. Namely, a 2-fold increase was observed after a 24h exposure, at 37° C, suggesting that PARP activation could be linked to an early adaptive response of cultured rat hepatocytes to simulated microgravity. Moreover, after a 24 h-exposure to simulated µG, cultured cells revealed a net inhibition of Topo I DNA relaxating activity, as compared to respective ground-based controls (-67%). This drop can be directly correlated to the increased PARP-1 activity observed on the same batch of cells.

THE MOTIVE FORCES OF LIGHT IN ADVANCED THERMODYNAMICS OF LIQUIDS

A. Sukhodolsky
General Physics Institute Russian Academy of Sciences, 38 Vavilov Street, Moscow, Russia

The purpose of this talk is to present the advanced principles to create the thermomotive forces of light by continuous optical pumping of the first order phase transitions in multiphase liquids and the outlook for the novel trends in chemical and mechanical engineering available by the direct thermo dynamic conversion of solar light in space.
THEORY: The gigantic thermomotive forces have been found in several new phenomena of laser physics, when direct generation of mechanical energy takes place in absorbing liquids, which play a role in the active work bodies by continuous optical pumping. These forces appear to be in many orders of magnitude bigger than radiation pressure of light can provide with. The theory of motive forces and the problem of maximum light available for direct conversion into mechanical energy are based on the application of entropy of Plank for consideration of creation and dissipation of optically pumped heat sources in liquids. The difference between non-equilibrium entropy of Plank for motive forces of light and equilibrium entropy of Clausius for motive forces of heat is discussed on the fundamental level. The first and second laws of the motives of thermodynamics are presented. As far as mechanical energy is involved and taken into consideration, the work for motive forces is supposed to consider in the complex number format to keep both the laws of energy and momentum conservation for any elementary source of mechanical energy created by light in multiphase liquids.
EXPERIMENT: A new state of liquid (inversion) to directly generate the periodical sequence of hydraulic shocks (10-2000HZ) under continuous wave (CW) laser optical pumping has been found. The power of each pulse is about 10 000 times bigger than the average power of pumping light with energy efficiency up to 30% estimated for solar pumping. Direct generation of propulsive energy by CW-radiation was used for several new technologies of pumping liquid, attracting gas bubbles, propelling solid bodies, processing transparent materials oriented on the energy-saving use of sunlight. The effect to attract opaque drops in transparent liquid by CW-light with velocity about 10cm/sec that is due to capillary forces at surface of phase discontinuity has been discovered. The phenomenon is explained to be due to the self organization of the non-equilibrium Gibbs surface. The new technology of liquid extraction based on motive forces of light in liquids is proposed. The working model of the capillary light-extractor was used for the separation of clean water from its mixture with an absorbing dye.


A MODEL OF OXYGEN TRANSPORT AS A TOOL TO STUDY THE LIQUID METAL SURFACE OXIDATION

E.Arato, M.Ratto &, E.Ricci*, R.Novakovic*, L.Fiori, D.Giuranno* and A.Passerone*
DIAM, Università di Genova, via dell'Opera Pia ,15 1-16125 Genova, Italy & Institute for Systems Informatics and Safety, JRC - E. C. TP 361, 21020 Ispra (VA), Italy *ICFAM -CNR, Via De Marini, 6 16149 Genova, Italy

The technological implications of the Marangoni effects are vast and very important: they span from chemical engineering to metallurgy, and from biology to crystal growth processes. In this particular field, the strong influence of oxygen partial pressure on the Marangoni flow of molten silicon has been recently demonstrated. In fact Silicon crystal growth technologies are controlled by complex heat and mass transfer processes which include buoyancy convection and Marangoni flow: the latter being one of the main causes of oxygen transfer into the crystal. As the driving force for the Marangoni flow is the surface tension gradient, the influence of temperature and adsorption parameters on the surface tension of molten silicon have to be known. Several studies have been made to explain the mechanism of oxygen adsorption at the liquid metals interface or to evaluate the actual oxygen concentration near the surface of the liquid metal. In fact, even today, when the surface tension of a liquid metal is measured as a function of oxygen concentration, one of the critical points of the experiments is to know exactly the oxygen equilibrium concentration in the bulk phase. This can be done either by a direct measurement inside the bulk liquid, or by deriving this quantity from the knowledge of the actual oxygen concentration near the surface of the liquid metal. The former solution is difficult to implement in surface properties measurements, usually made with small quantities of the liquid metal. The latter requires a full knowledge of the thermodynamics, kinetics and the fluid dynamics of the system. The theoretical analysis presented here allows for the behaviour of molten metals in the presence of oxygen to be described.
A generalised Wagner approach has been adopted for molten metals, forming volatile oxides, in which the description of oxygen transfer from the gas phase to the condensed phase must account for the double contribution of molecular oxygen and oxygen linked as oxide, which leads to define the concept of 'Oxygen Effective Pressure'. One of the hypotheses of the model is the complete absence of buoyancy-driven convection making it appealing for verification under microgravity conditions. From the analysis of the system at varying operating conditions, it was possible to relate the gas phase composition at the surface, which is hardly measurable, to the composition in the feed or at the outlet.
Following the Si-O system thermodynamic analysis, previously presented at the ELGRA meeting held in Rome in 1999, the application of the developed theory to the molten silicon-oxygen system is presented here. The effectiveness of such a theory as a supporting tool for experimental work of crystal growth processes is discussed and verified.

Interdiffusion Experiment in liquid PdNiP and PdCuNiP under 1g conditionS

S.Suzuki, M.-P.Macht*, A.Griesche, K.-H.Kraatz and G.Frohberg
Institute for Metallic Materials, Technical University of Berlin, Secr.PN 2-3, Hardenbergstr. 36, D-10623 Berlin, Germany *Hahn-Meitner-Institut Berlin, Glienicker Str. 100, D-14109 Berlin, Germany

The alloys PdNiP and PdCuNiP form stable metallic glasses and have highly viscous liquid melts. Thus diffusion experiments of comparatively long duration in the amorphous phase, in the supercooled liquid state, in the undercooled liquid melt and in the liquid melt allow better understanding of the diffusion behavior in the transient range between liquid and solid, which may give deeper insight in the mass transport which controls the crystallization.
As a reference measurement for a planned microgravity experiment an interdiffusion experiment between liquid PdNiP and PdCuNiP under 1 g conditions was performed at 1123 K (239K above the melting point of PdNiP) by use of the long capillary method. After solidification of the diffusion couple with a cooling rate of 0.6 K/s some different crystalline phases with grain sizes up to 100m were detected. Longitudinal concentration profiles were determined by use of Energy-Dispersive X-Ray Spectrometry. From these concentration profiles diffusion coefficients of about 2 x10^-10 m²/s were obtained. This value corresponds to a result obtained from quasi-elastic neutron scattering measurements in liquid PdCuNiP-melts.

REMOTE ACCESS TO LIFE SCIENCES EXPERIMENTS ON THE ISS OR IN GROUND – BASED FACILITIES DEMONSTRATION OF A USER HOME BASE

M. Schuber, D. Seibt and P. Esser
DLR Microgravity User Support Center, Linder Hoehe 23b, D-51147 Köln

Europe voted for a decentralized support of the Columbus facilities involving Facility Responsible Centers and Facility Support Centers. User Home Bases will enable to access scientific data at the user`s home lab via a remote link. By this approach scientists need not be hosted at a Control Center to follow multiple or long-duration life sciences experiments in ISS facilities, e.g. with the BIOLAB. Moreover scientific–technical ground infrastructure and tools can be accessed by the scientists from their home labs. Being supplied with recent experimental data from BIOLAB or from ground-based facilities the experimenter can focus on research activities in the home lab. No duplication of this infrastructure will be necessary. Specific tools can be developed and adapted in accordance with the scientific requirements for processing and evaluation of experimental data.
The demonstration during the ELGRA meeting makes use of the lab model of the Slow Rotating Centrifuge Microscope NIZEMI investigating biosamples (sea urchin or ciliated protozoa) at the DLR Microgravity User Support Center in Köln. Remote access will be shown by processing of real-time video images for experiment evaluation.

THE CONTROL AND POWER SYSTEM OF THE MATERIALS SCIENCES LABORATORY FOR DESTINYK.

Kemmerle, P. Rank and U. Brammer
Kayser-Threde GmbH Perchtingerstr. 3 D-81379 München

The Control and Power System of the Materials Science Laboratory for Destiny is under responsibility of Kayser-Threde. The actual status is: the EM and the two Science Reference Models are manufactured and integrated. The environmental tests with the EM have been passed successfully. The functional performance test is under preparation and will be performed in August, followed by delivery to the rack integrator. The system is set-up as two separate rack drawers, Facility Control Unit and Power Supply Unit. Both units are controlled by an own controller, based on the Standard Payload Computer (SPLC) out of the Standard Payload Outfitting Equipment (SPOE) program of ESA. The analogue signal conditioning modules including the thermocouple amplifiers and all other analogue or digital control modules have been developed especially for MSL. Altogether there are nine modules for signal conditioning and sub-unit control, consisting of base board and four Mezzanine modules each. Six out of these 36 Mezzanine positions are spare and might be used for further upgrading of the system. The power supply for the heaters has been set-up as a modular system of 12 identical heater current sources for 300 W each (max. envelope: 40 V, 7.5 A each) appropriate for parallel and serial operation. In addition there are a 13th heater current source for sample reservoir heating and two Peltier current sources with a maximum current of 50 A each, also appropriate for parallel or serial operation. There are wired spare slots for two further heater current sources. Besides these units, the Quench Drive Electronics and the Magnetic Field Generator are accommodated in the Power Supply Unit. Block diagrams, photos and essential technical data will be presented in a poster at the meeting. The two subsystems have been set-up in a consequent modular manner such that in-orbit maintenance (e.g. exchange of failed modules or exchange against other modules with other functionality) will be possible. Re-programming capability is given, too.